Note: Descriptions are shown in the official language in which they were submitted.
CA 02949315 2016-11-23
RNASE H-BASED ASSAYS UTILIZING MODIFIED RNA MONOMERS
FIELD OF THE INVENTION
10021 This invention pertains to methods of cleaving a nucleic acid strand
to
initiate, assist, monitor or perform biological assays.
BACKGROUND OF THE INVENTION
[003] The specificity of primer-based amplification reactions, such as the
polymerase chain reaction (PCR), largely depends on the specificity of primer
hybridization with a DNA template. Under the elevated temperatures used in a
typical
amplification reaction, the primers ideally hybridize only to the intended
target
sequence and form primer extension products to produce the complement of the
target
sequence. However, amplification reaction mixtures are typically assembled at
room
temperature, well below the temperature needed to insure primer hybridization
specificity. Under lower temperature conditions, the primers may bind non-
specifically
to other partially complementary nucleic acid sequences or to other primers
and initiate
the synthesis of undesired extension products, which can be amplified along
with the
target sequence. Amplification of non-specific primer extension products can
compete
with amplification of the desired target sequences and can significantly
decrease the
efficiency of the amplification of the desired sequence. Non-specific
amplification can
also give rise in certain assays to a false positive result.
[004] One frequently observed type of non-specific amplification product in
PCR
is a template independent artifact of the amplification reaction known as
"primer
dimers". Primer dimers are double-stranded fragments whose length typically is
close
to the sum of the two primer lengths and are amplified when one primer is
extended
over another primer. The resulting duplex forms an undesired template which,
because
of its short length, is amplified efficiently.
[005] Non-specific amplification can be reduced by reducing the formation
of
primer extension products (e.g., primer dimers) prior to the start of the
reaction. In one
method, referred to as a "hot-start" protocol, one or more critical reagents
arc withheld
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CA 02949315 2016-11-23
from the reaction mixture until the temperature is raised sufficiently to
provide the
necessary hybridization specificity. In this manner, the reaction mixture
cannot support
primer extension at lower temperatures. Manual hot-start methods, in which the
reaction tubes are opened after the initial high temperature incubation step
and the
missing reagents are added, are labor intensive and increase the risk of
contamination
of the reaction mixture.
[0061 Alternatively, a heat sensitive material, such as wax, can be used to
separate
or sequester reaction components, as described in U.S. Pat. No. 5,411,876, and
Chou et
al., 1992, Nucl. Acids Res. 20(7):1717-1723. In these methods, a high
temperature
pre-reaction incubation melts the heat sensitive material, thereby allowing
the reagents
to mix.
[007] Another method of reducing the formation of primer extension products
prior to the start of PCR relies on the heat-reversible inactivation of the
DNA
polymerase. U.S. Pat. Nos. 5,773,258 and 5,677,152,
describe DNA polymerases reversibly inactivated by the covalent
attachment of a modifier group. Incubation of the inactivated DNA polymerase
at high
temperature results in cleavage of the modifier-enzyme bond, thereby releasing
an
active form of the enzyme. Non-covalent reversible inhibition of a DNA
polymerase
by DNA polymerase-specific antibodies is described in U.S. Pat. Nos.
5,338,671.
[0081 One objective of the present invention can be used, for example, to
address
the problem of carry-over cross contamination which is a significant concern
in
amplification reactions, especially PCR wherein a large number of copies of
the
amplified product are produced. In the prior art, attempts have been made to
solve this
problem in a number of ways. For example, direct UV irradiation can
effectively
remove contaminating DNA (Rys & Persing, 1993, J Clin Microbiol. 31(9):2356-60
and Sarkar & Sommer, 1990 Nature. 343(6253):27) but the irradiation of the PCR
reagents must take place before addition of polymerase, primers, and template
DNA.
Furthermore, this approach may be inefficient because the large numbers of
mononucleotides present in the reaction will absorb much of the UV light. An
alternative, the "UNG method", incorporates dUTP into the amplified fragments
to
alter the composition of the product so that it is different from native,
naturally
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CA 02949315 2016-11-23
occurring DNA (Longo et al. 1990, Gene, 93(1): 125-128). The enzyme
Uracil-N-Glycosylase (UNG) is added together with the other components of the
PCR
mixture. The UNG enzyme will cleave the uracil base from DNA strands of
contaminating amplicons before amplification, and render all such products
unable to
act as a template for ncvvr DNA synthesis without affecting the sample DNA.
The LNG
enzyme is then heat-inactivated and PCR is then carried out. The requirement
for
dUTP and the UNG enzyme adds significantly to the cost of performing PCR.
[009] Another objective of the present invention is to provide PCR assays
in
which a hot-start reaction is achieved through a coupled reaction sequence
with a
thermostable RNase H.
Ribonuclease Enzymes
[010] Ribonucleases (RNases) are enzymes that catalyze the hydrolysis of
RNA
into smaller components. The enzymes are present internally; in bodily fluids;
on the
surface of skin; and on the surface of many objects, including untreated
laboratory
glasswear. Double-stranded RNases are present in nearly all intracellular
environments
and cleave RNA-containing, double-stranded constructs. Single-stranded RNases
are
ubiquitous in extracellular environments, and are therefore extremely stable
in order to
function under a wide range of conditions.
[011] The RNases H are a conserved family of ribonucleases which are
present in
all organisms examined to date. There are two primary classes of RNase H:
RNase H1
and RNase H2. Retroviral RNase H enzymes are similar to the prokaryotic RNase
HI.
All of these enzymes share the characteristic that they are able to cleave the
RNA
component of an RNA:DNA heteroduplex. The human and mouse RNase H1 genes are
78% identical at the amino acid level (Cerritelli, et al., (1998) Genomics,
53, 300-307).
In prokaryotes, the genes are named rnha (RNase H1) and rnhb (RNase H2). A
third
family of prokaryotic RNases has been proposed, rnhc (RNase H3) (Ohtani, et
al. (1999)
Biochemistry, 38, 605-618).
[012] Evolutionarily, "ancient" organisms (archaeal species) in some cases
appear
to have only a single RNase H enzyme which is most closely related to the
modern
RNase H2 enzymes (prokaryotic) (Ohtani, et al., .1 Biosci Bioeng, 88, 12-19).
Exceptions do exist, and the archaeal Halobacterium has an RNase HI ortholog
(Ohtani,
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CA 02949315 2016-11-23
et al., (2004) Biochem J, 381, 795-802). An RNase H1 gene has also been
identified in
Thermus thermophilus (Itaya, et al., (1991) Nucleic Acids Res, 19, 4443-4449).
RNase
H2 enzymes appear to be present in all living organisms. Although all classes
of RNase
H enzymes hydrolyze the RNA component of an RNA:DNA heteroduplex, the
substrate and co-factor requirements are different. For example, the Type II
enzymes
utilize Mg¨, Mn¨ , Co-' (and sometimes Ni) as cofactor, while the Type I
enzymes
require Mg and can be inhibited by Mn' ions. The reaction products are the
same for
both classes of enzymes: the cleaved products have a 3'-OH and 5'-phosphate
(See
Figure 1). RNase ITT class enzymes which cleave RNA:RNA duplexes (e.g., Dicer,
Ago2, Drosha) result in similar products and contain a nuclease domain with
similarity
to RNase H. Most other ribonucleases, and in particular single stranded
ribonucleases,
result in a cyclic 2',3'-phosphate and 5'-OH products (see Figure 2).
Type 1 RNase H
10131 E. coli RNase HI has been extensively characterized. A large amount
of
work on this enzyme has been carried out, focusing on characterization of
substrate
requirements as it impacts antisense oligonucleotide design; this has included
studies
on both the E . coil RNase H1 (see Crooke, et al., (1995) B iochetn J, 312 (Pt
2), 599-608;
Lima, et al., (1997)1 Biol Chem, 272, 27513-27516; Lima, et al., (1997)
Biochemistry,
36, 390-398; Lima, et al., (1997) J Biol Chem, 272, 18191-18199; Lima, et al.,
(2007)
Mol Phannacol, 71, 83-91; Lima, et al., (2007) Mol Pharmacol, 71, 73-82; Lima,
et al.,
(2003) 1 Biol Chem, 278, 14906-14912; Lima, et al., (2003) J Biol Chem, 278,
49860-49867) and the Human RNase HI (see Wu, et al., (1998)Antisense Nucleic
Acid
Drug Dev, 8, 53-61; Wu, et al., (1999) J Biol Chem, 274, 28270-28278; Wu, et
al.,
(2001) J Biol Chem, 276, 23547-23553). In tissue culture, overexpression of
human
RNase HI increases potency of antisense oligos (AS0s) while knockdown of RNase
H1 using either siRNAs or ASOs decreases potency of antisense
oligonucleotides.
10141 Type I RNase H requires multiple RNA bases in the substrate for full
activity. A DNA/RNA/DNA oligonucicotide (hybridized to a DNA oligonucleotidc)
with only 1 or 2 RNA bases is inactive. With E. coli RNase H1 substrates with
three
consecutive RNA bases show weak activity. Full activity was observed with a
stretch
of four RNA bases (Hogrefe, et al., (1990) 1 Biol Chem, 265, 5561-5566). An
RNase
HI was cloned from Therm us thermophilus in 1991 which has only 56% amino acid
4
CA 02949315 2016-11-23
identity with the E. coli enzyme but which has similar catalytic properties
(Itaya, et al.,
(1991) Nucleic Acids Res, 19, 4443-4449). This enzyme was stable at 65 C but
rapidly
lost activity when heated to 80 C.
[015] The human RNase HI gene (Type I RNase H) was cloned in 1998
(Genomics, 53, 300-307 and Antisense Nucleic Acid Drug Dev, 8, 53-61). This
enzyme
requires a 5 base RNA stretch in DNA/RNA/DNA chimeras for cleavage to occur.
Maximal activity was observed in I mM Mg buffer at neutral pH and Mn+-' ions
were
inhibitory (J Biol Chem, 274, 28270-28278). Cleavage was not observed when
2'-modified nucleosides (such as 2'-0Me, 2'-F, etc.) were substituted for RNA.
10161 Three amino acids (Asp-10, Glu-48, and Asp-70) make up the catalytic
site
of E. coli RNase HI which resides in the highly conserved carboxy-terminal
domain of
the protein (Katayanagi, et al., (1990) Nature, 347, 306-309); this domain has
been
evaluated by both site directed mutagenesis and crystal structure
determination. The
same amino acids are involved in coordination of the divalent ion cofactor.
10171 Interestingly, 2'-modification of the substrate duplex alters the
geometry of
the helix and can adversely affect activity of RNase Hi. 2'- 0 -(2-
methoxy)ethyl
(MOE) modifications flanking the RNA segment reduce cleavage rates, presumably
due to alterations in the sugar conformation and helical geometry. Locked
nucleic acid
(LNA) bases perturb helical geometry to a greater degree and impacted enzyme
activity
to a greater extent (Mol Phartnacol, 71, 83-91 and Mol Phartnacol, 71, 73-82).
Damha
(McGill University) has studied the effects of 2'-F modified nucleosides
(2'-deoxy-2'-fluoro-b-D-ribose) when present in the substrate duplex and finds
that this
group cannot be cleaved by RNase HI (Yazbeck, et al., (2002) Nucleic Acids
Res, 30,
3015-3025). Formulas A and B illustrate the two different mechanisms that have
been
proposed for RNase HI cleavage, both of which require participation of the
2'0H
group.
CA 02949315 2016-11-23
A
=0 fa
.õ.,
'!"?7 __________________ .11/ *.;kit,,Fmarariefexami
4 ¨
= .fte
, I
6
õ.
a".
Formulas A and B
[018] Damha's studies are consistent with the known active site of the
enzyme,
wherein the reaction mechanism involves the 2'-OH group. The enzyme active
site
resides within a cluster of lysine residues which presumably contribute to
electrostatic
binding of the duplex. Interaction between the binding surface and negatively
charged
phosphate backbone is believed to occur along the minor grove of the RNA:DNA
heteroduplex (Nakamura, et al., (1991) Proc Nati Acad Sci USA, 88, 11535-
11539);
changes in structure that affect the minor groove should therefore affect
interactions
between the substrate and the active site. For example, the minor groove width
is 7.5 A
in a B-form DNA:DNA duplex, is II A in a pure A-form RNA:RNA duplex, and is
8.5
A in the hybrid A-form duplex of an RNA:DNA duplex (Fedoroff et al., (1993)J
Mol
ho!, 233, 509-523). 2'-modifications protrude into the minor groove, which may
account for some of the behavior of these groups in reducing or eliminating
activity of
modified substrates for cleavage by RNase Hi. Even a 2'-F nucleoside, which is
the
most "conservative" RNA analog with respect to changing chemical structure,
adversely affects activity.
Type II RNase H
[019] The human Type II RNase H was first purified and characterized by
Eder
and Walder in 1991 (Eder, et al., (1991)i Biol Chem, 266, 6472-6479). This
enzyme
was initially designated human RNase HI because it had the characteristic
divalent
metal ion dependence of what was then known as Class I RNases H. In the
current
6
CA 02949315 2016-11-23
nomenclature, it is a Type II RNase H enzyme. Unlike the Type I enzymes which
are
active in Mg +- but inhibited by Mn¨ ions, the Type Il enzymes are active with
a wide
variety of divalent cations. Optimal activity of human Type II RNase H is
observed
with 10 mM Mg' 5 mM Co' `, or 0.5 mM Mn.
10201 Importantly, the substrate specificity of the Type II RNase H
(hereafter
referred to as RNase H2) is different from RNase Hi. In particular, this
enzyme can
cleave a single ribonucleotide embedded within a DNA sequence (in duplex form)
(Eder, et al., (1993) Biochimie, 75, 123-126). Interestingly, cleavage occurs
on the 5'
side of the RNA residue (See Figure 3). See a recent review by Kanaya for a
summary
of prokaryotic RNase H2 enzymes (Kanaya (2001) Methods Enzymol, 341, 377-394).
10211 The E. coli RNase H2 gene has been cloned (Itaya, M. (1990) Proc Nat!
Acad Sci US A, 87, 8587-8591) and characterized (Ohtani, et al., (2000) J
Biochem
(Tokyo), 127, 895-899). Like the human enzyme, the E. coli enzyme functions
with
Mn¨ ions and is actually more active with manganese than magnesium.
[022] RNase H2 genes have been cloned and the enzymes characterized from a
variety of eukaryotic and prokaryotic sources. The RNase H2 from Pyrococcus
kodakaraensis (KOD1) has been cloned and studied in detail (Haruki, et al.,
(1998) J
Bacteriol, 180, 6207-6214; Mukaiyama, etal., (2004) Biochemistry, 43, 13859-
13866).
The RNase H2 from the related organism Pyrococcus furious has also been cloned
but
has not been as thoroughly characterized (Sato, et al., (2003) Biochem Biophys
Res
Commun, 309, 247-252).
10231 The RNasc H2 from Methanococcus jannaschii was cloned and
characterized by Lai (Lai, et al., (2000) Structure, 8, 897-904; Lai et al.,
(2003)
Biochemistry, 42, 785-791). Isothermal titration calorimetry was used to
quantitatively
measure metal ion binding to the enzyme. They tested binding of Mn, Mg+' ,
Ca++,
and Bail and in all cases observed a 1:1 molar binding ratio, suggesting the
presence of
only a single divalent metal ion cofactor in the enzyme's active site. The
association
constant for Mn was 10-fold higher than for Mg'''. Peak enzyme activity was
seen at
0.8 mM MnC12.
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CA 02949315 2016-11-23
10241 Nucleic acid hybridization assays based on cleavage of an RNA-
containing
probe by RNase H such as the cycling probe reaction (Walder et al., U.S. Pat.
No.
5,403,711) have been limited in the past by background cleavage of the
oligonucleotide
by contaminating single-stranded ribonucleases and by water catalyzed
hydrolysis
facilitated by Mgn and other divalent cations. The effect of single-stranded
ribonucleases can be mitigated to a certain degree by inhibitors such as
RNasin that
block single-stranded ribonucleases but do not interfere with the activity of
RNase H.
10251 Single-stranded ribonucleases cleave 3' of an RNA residue, leaving a
cyclic
phosphate group at the 2' and 3' positions of the ribose (See FIG. 2). The
same
products are produced by spontaneous water catalyzed hydrolysis. In both
cases, the
cyclic phosphate can hydrolyze further forming a 3'-monophosphate ester in the
enzyme catalyzed reaction, or a mixture of the 3'- and 2' -monophosphate
esters
through spontaneous hydrolysis. The difference between the cleavage products
formed
by RNase H (FIG. 1) and those formed by nonspecific cleavage of the probe
(FIG. 2)
provides a basis for distinguishing between the two pathways. This difference
is even
more pronounced when comparing cleavage by RNase H2 and single-stranded
ribonucleases with substrates having only a single RNA residue. In that case,
RNase
H2 and single-stranded ribonucleases attack at different positions along the
phosphate
backbone (See Figure 3).
10261 RNase H has been used as a cleaving enzyme in cycling probe assays,
in
PCR assays (Han et al., U.S. Patent No. 5,763,181; Sagawa et al., U.S. Pat.
No.
7,135,291; and Behlke and Walder, U.S. Pat. App. No. 20080068643) and in
polynomial amplification reactions (Behlke etal., U.S. Patent No. 7,112,406).
Despite
improvements offered by these assays, there remain considerable limitations.
The PCR
assays utilize a hot-start DNA polymerase which adds substantially to the
cost.
Moreover, each time an alternative DNA polymerase is required a new hot-start
version
of the enzyme must be developed. In addition, the utility of these various
assays has
been limited by undesirable cleavage of the oligonucleotide probe or primer
used in the
reaction, including water and divalent metal ion catalyzed hydrolysis 3' to
RNA
residues, hydrolysis by single-stranded ribonucleases and atypical cleavage
reactions
catalyzed by Type II RNase H enzymes at positions other than the 5'-phosphate
of an
8
CA 02949315 2016-11-23
RNA residue. The present invention overcomes these limitations and offers
further
advantages and new assay formats for use of RNase H in biological assays.
[0271 The current
invention provides novel biological assays that employ RNase
H cleavage in relation to nucleic acid amplification, detection, ligation,
sequencing, and
synthesis. Additionally, the invention provides new assay formats to utilize
cleavage
by RNasc H and novel oligonucicotide substrates for such assays. The
compounds, kits,
and methods of the present invention provide a convenient and economic means
of
achieving highly specific primer-based amplification reactions that are
substantially
free of nonspecific amplification impurities such as primer dimers. The
methods and
kits of the present invention avoid the need for reversibly inactivated DNA
polymerase
and DNA ligase enzymes.
BRIEF SUMMARY OF THE INVENTION
[0281 One objective
of the present invention is to enable hot start protocols in
nucleic acid amplification and detection assays including but not limited to
PCR, OLA
(oligonucleotide ligation assays), LCR (ligation chain reaction), polynomial
amplification and DNA sequencing, wherein the hot start component is a
thermostable
RNase H or other nicking enzyme that gains activity at the elevated
temperatures
employed in the reaction. Such assays employ a modified oligonucleotide of the
invention that is unable to participate in the reaction until it hybridizes to
a
complementary nucleic acid sequence and is cleaved to generate a functional 5'-
or
3' -end. Compared to the corresponding assays in which standard unmodified DNA
oligonucleotides are used the specificity is greatly enhanced. Moreover the
requirement for reversibly inactivated DNA polymerases or DNA ligases is
eliminated.
[029] In the case of
assays involving primer extension (e.g., PCR, polynomial
amplification and DNA sequencing) the modification of the oligonucleotide
inhibiting
activity is preferably located at or near the 3'-end. In some embodiments
where the
oligonucleotides are being used as primers, the oligonucleotide inhibiting
activity may
be positioned near the 3' end of the oligonucleotide, e.g., up to about 10
bases from the
3' end of the oligonucleotide of the invention. In other embodiments, the
oligonucleotide inhibiting activity may be positioned near the 3' end, e.g.,
about 1-6
bases from the 3' end of the oligonucleotide of the invention. In other
embodiments,
9
CA 02949315 2016-11-23
the oligonucleotide inhibiting activity may be positioned near the 3' end,
e.g., about 1-5
bases from the 3' end of the oligonucleotide of the invention. In other
embodiments, the
oligonucleotide inhibiting activity may be positioned near the 3' end, e.g.,
about 1-3
bases from the 3' end of the oligonucleotide of the invention. In other
embodiments,
the precise position (i.e., number of bases) from the 3' end where the
oligonucleotide
inhibiting activity may be positioned will depend upon factors influencing the
ability of
the oligonucleotide primer of the invention to hybridize to a shortened
complement of
itself on the target sequence (i.e., the sequence for which hybridization is
desired). Such factors include but arc not limited to Tm, buffer composition,
and
annealing temperature employed in the reaction(s).
1030] For ligation assays (e.g., OLA and LCR) the modification inhibiting
activity
may be located at or near either the 3'- or 5'-end of the oligonucleotide. In
other
embodiments, for ligation assays, modification inhibitory activity, if used,
is preferably
placed within the domain that is 3' to the cleavable RNA base in the region
that is
removed by probe cleavage. In other embodiments, for ligation assays, C3
spacers may
be positioned close to the RNA base in the oligonucleotide probes of the
invention to
improve specificity that is helpful for improving mismatch discrimination. In
other
embodiments, in an OLA assay, where readout depends upon a PCR assay to
amplify
the product of a ligation event, any blocking group may be placed in the
domain of the
oligonucleotide of the invention that is removed by RNase H cleavage. In such
embodiments, in an OLA assay where readout depends upon a PCR assay to amplify
the product of a ligation event, the precise position of the blocking group in
the RNase
H cleavable domain may be adjusted to alter specificity for cleavage and
precise
placement of the blocking group relative to the cleavable RNA bases may alter
the
amount of enzyme needed to achieve optimal cleavage rates.
10311 Yet a further objective of the present invention is to provide novel
modifications of oligonucleotides to interfere with primer extension and
ligation.
[032] Yet a further objective of the present invention is to provide
modifications
of oligonucleotides that prevent the oligonucleotide from serving as a
template for
DNA synthesis and thereby interfere with PCR.
CA 02949315 2016-11-23
[033] Yet a further objective of the invention is to provide modified
oligonucleotide sequences lacking RNA that are cleaved by RNase H. In one such
embodiment, the oligonucleotide contains a single 2'-fluoro residue and
cleavage is
mediated by a Type II RNase H enzyme. In a more preferred embodiment the
oligonucleotide contains two adjacent 2'-fluoro residues.
[034] Yet a further objective of the present invention is to provide
oligonucleotides for use in the above mentioned assays that are modified so as
to inhibit
undesired cleavage reactions including but not limited to water and divalent
metal ion
catalyzed hydrolysis 3' to RNA residues, hydrolysis by single-stranded
ribonucleases
and atypical cleavage reactions catalyzed by Type II RNase H enzymes at
positions
other than the 5'-phosphate of an RNA residue (see Figure 3). In one such
embodiment
the 2'-hydroxy group of an RNA residue is replaced with an alternative
functional
group such as fluorine or an alkoxy substituent (e.g., 0-methyl). In another
such
embodiment the phosphate group 3' to an RNA residue is replaced with a
phosphorothioate or a dithioate linkage. In yet another embodiment the
oligonucleotidc
is modified with nuclease resistant linkages further downstream from the 3'-
phosphate
group of an RNA residue or on the 5'-side of an RNA residue to prevent
aberrant
cleavage by RNase H2. Nuclease resistant linkages useful in such embodiments
include phosphorothioates, dithioates, methylphosphonates, and abasic residues
such
as a C3 spacer. Incorporation of such nuclease resistant linkages into
oligonucleotide
primers used in PCR assays of the present invention has been found to be
particularly
beneficial (see Examples 25, 27 and 28 ).
[035] Yet a further objective of the invention is to provide
oligonucleotides for
use in the above-mentioned assays that are modified at positions flanking the
cleavage
site to provide enhanced discrimination of variant alleles. Such modifications
include
but are not limited to 2'-0-methyl RNA residues and secondary mismatch
substitutions
(see Example 23).
10361 Yet a further objective is to provide oligonucleotides and assay
formats for
use in the present invention wherein cleavage of the oligonucleotide can be
measured
by a change in fluorescence. In one such embodiment a primer cleavable by
RNase H is
labeled with a fluorophore and a quencher and the assay is monitored by an
increase in
fluorescence (see Examples 19-21).
11
CA 02949315 2016-11-23
[037] Yet a further objective of the invention is to provide RNase H
compositions
and protocols for their use in which the enzyme is thermostable and has
reduced
activity at lower temperatures.
[038] In yet a further embodiment a Type II RNase H is employed in a
cycling
probe reaction in which the RNA residue in the probe is replaced with a 2'-
fluoro
residue. In a more preferred embodiment a probe with two adjacent 2'-fluoro
residues
is used.
10391 Many of the aspects of the present invention relating to primer
extension
assays, ligation assays and cycling probe reactions are summarized in Tables
1, 2, and 3,
respectively.
[040] In yet a further embodiment of the invention Type II RNasc H enzymes
are
used in novel methods for DNA sequencing.
[041] In yet a further embodiment of the invention Type II RNase H enzymes
are
used in novel methods for DNA synthesis.
BRIEF DESCRIPTION OF THE FIGURES
[042] Figure 1 depicts the cleavage pattern that occurs with an RNase H
enzyme
on a substrate containing multiple RNA bases.
[043] Figure 2 depicts the cleavage pattern that occurs with a single-
stranded
ribonucleasc enzyme or through water catalyzed hydrolysis, wherein the end-
product
results in a cyclic phosphate group at the 2' and 3' positions of the ribose.
[044] Figure 3 depicts the cleavage sites for RNase H2 and single-stranded
ribonucleases on a substrate containing a single RNA base.
[045] Figures 4A and 4B arc photographs of SDS 10% polyacrylamide gels that
illustrate the induced protein produced from five Archaeal RNase H2 synthetic
genes.
Figure 4A shows induced protein for Pyrococcus furiosus and Pyrococus abyssi.
Figure 4B shows induced protein for Methanocaldococcus jannaschii, Sulfolobus
solfataricus, Pyrococcus kodadarensis.
12
CA 02949315 2016-11-23
[046] Figure 5 shows a Coomassie Blue stained protein gel showing pure,
single
bands after purification using nickel affinity chromatography of recombinant
His tag
RNase H2 proteins.
[047] Figure 6 shows a Western blot done using anti-His tag antibodies
using the
protein gel from FIG. 5.
[048] Figure 7 is a photograph of a gel that shows the digestion of a
duplex,
containing a chimeric 11 DNA ¨ 8 RNA ¨ 11 DNA strand and a complementary DNA
strand, by recombinant RNasc H2 enzymes from Pyrococcus kodakaraensis,
Pyrococcus furiosus, and Pyrococcus abyssi.
[049] Figures 8A and 8B are photographs of gels that show the digestion of
a
duplex, containing a chimeric 14 DNA ¨ 1 RNA ¨ 15 DNA strand and a
complementary DNA strand, by recombinant RNase H2 enzymes from Pyrococcus
abyss", Pyrococcus jitriosus, and Methanocaldococcus jannaschii (FIG. 8A) and
Pyrococcus kodakaraensis (FIG. 8B).
[050] Figure 9 shows the effects of incubation at 95 C for various times
on the
activity of the Pyrococcus abyssi RNase H2 enzyme.
[051] Figure 10 is a photograph of a gel that shows the relative amounts of
cleavage of a single ribonucleotide-containing substrate by Pyrococcus abyssi
RNase
H2 at various incubation temperatures.
[052] Figure 11 is a graph showing the actual quantity of substrate cleaved
in the
gel from FIG. 10.
[053] Figure 12 is a photograph of a gel that shows cleavage by Pyrococcus
abysii
RNase H2 of various single 2' modified substrates in the presence of different
divalent
cations.
[054] Figure 13 is a photograph of a gel that shows cleavage by Pyrococcus
abys.si
RNase H2 of single 2'-fluoro or double 2'-fluoro (di-fluoro) modified
substrates. The
divalent cation present was Mn-f.
13
CA 02949315 2016-11-23
[055] Figure 14 is a graph quantifying the relative cleavage by Pyrococcus
abyssi
RNase H2 of all 16 possible di-fluoro modified substrates.
[056] Figure 15 is a graph quantifying the relative cleavage by Pyrococcus
abyssi
RNase H2 of rN substrates with a variable number of 3' DNA bases (i.e., number
of
DNA bases on the 3' side of the RNA residue).
[057] Figure 16 is a graph quantifying the relative cleavage by Pyrococcus
abyssi
RNase H2 of rN substrates with a variable number of 5' DNA bases (i.e., number
of
DNA bases on the 5' side of the RNA residue).
[058] Figure 17 is a graph quantifying the relative cleavage by Pyrococcus
abyssi
RNase H2 of di-fluoro substrates with a variable number of 3' DNA bases (i.e.,
number
of DNA bases on the 3' side of the fUfC residues).
10591 Figure 18 is a reaction schematic of RNase H2 activation of blocked
PCR
primers.
[060] Figure 19 is a photograph of a gel that shows the products of an end
point
PCR reaction performed with a single rU-containing blocked primer. The suffix
2D,
3D, etc. represents the number of DNA bases between the rU residues and the 3'-
end of
the primer. The primer is blocked with a dideoxy C residue.
[061] Figures 20A-B are PCR amplification plots for a 340 bp amplicon
within
the human HRAS gene, using both unmodified and blocked rN primers, without
RNase
H2 (20A) and with RNase H2 (20B). Cycle number is shown on the X-axis and
relative
fluorescence intensity is shown on the Y-axis.
[062] Figures 21A-B are PCR amplification plots for a 184 bp amplicon
within
the human ETS2 gene, using both unmodified and blocked rN primers, without
RNase
H2 (21A) and with RNase H2 (21B). Cycle number is shown on the X-axis and
relative
fluorescence intensity is shown on the Y-axis.
[063] Figures 22A-B are PCR amplification plots for a synthetic 103 bp
amplicon,
using both unmodified and 3'-fN modified primers, without RNase H2 (22A) and
with
RNase H2 (22B).
14
CA 02949315 2016-11-23
[064] Figure 23A shows HPLC traces of a rN primer containing a single
phosphorothioate intemucleoside modification. The top panel shows the original
synthesis product demonstrating resolution of the two isomers. The middle
panel is the
purified Rp isomer and the bottom panel is the purified Sp isomer.
10651 Figure 24 shows the relationship between RNase H2 versus RNase A
enzymatic cleavage with substrates having a single RNA base and different
phosphorothioate stereoisomers.
10661 Figure 25 shows a photograph of a polyacrylamide gel used to separate
products from PCR reactions done using standard and blocked/cleavable primers
on a
HCV amplicon showing that use of standard primers results in formation of
undesired
small primer-dimer species while use of blocked primers results in specific
amplification of the desired product. The nucleic acids were imaged using
fluorescent
staining and the image was inverted for clarity.
[067] Figure 26 is a graph quantifying the relative cleavage by Pyrococcus
abyssi
RNase H2 of a radiolabeled rC containing substrate in buffer containing
different
detergents at different concentrations (expressed as % vol:vol).
[068] Figure 27 is a reaction schematic of RNase H2 activation of
fluorescence-quenched (F/Q) blocked PCR primers.
[069] Figure 28 is an amplification plot showing the fluorescence signal
resulting
from use of unblocked primers with a fluorescence-quenched dual-labeled probe
(DLP)
compared with a blocked fluorescence-quenched cleavable primer for a 103 base
synthetic amplicon. Cycle number is shown on the X-axis and relative
fluorescence
intensity is shown on the Y-axis.
[070] Figure 29 is an amplification plot showing the fluorescence signal
resulting
from use of a F/Q configuration blocked fluorescence-quenched cleavable primer
compared with a Q/F configuration blocked fluorescence-quenched cleavable
primer
for a 103 base synthetic amplicon. Cycle number is shown on the X-axis and
relative
fluorescence intensity is shown on the Y-axis.
CA 02949315 2016-11-23
[071] Figure 30 is an amplification plot showing the fluorescence signal
resulting
from use of F/Q configuration blocked fluorescence-quenched cleavable primers
to
distinguish DNA templates that differ at a single base within the SMAD 7 gene.
Panel
(A) shows results from the FAM channel where the FAM-labeled "C" allele probe
was
employed. Panel "B" shows results from the HEX channel wherein the HEX-labeled
"T" allele probe was employed. Cycle number is shown on the X-axis and
relative
fluorescence intensity is shown on the Y-axis.
[072] Figure 31 is a
reaction schematic of RNase H2 cleavage of
fluorescence-quenched (FQT) primer used in a primer probe assay. The Primer
Domain is complementary to the target nucleic acid and serves to primer DNA
synthesis. The Reporter Domain is non-complementary to target and contains a
RNA
base positioned between a reporter dye and a quencher group. The Reporter
Domain
remains single-stranded until conversion to double-stranded form during PCR
where
this domain now serves as template. Conversion to double-stranded form
converts the
Reporter Domain into a substrate for RNase H2; cleavage by RNase H2 separates
reporter from quencher and is a detectable event.
[073] Figure 32 shows amplification plots of qPCR reactions done with
primers
specific for the human Drosha gene using HeLa cell cDNA. A) Reactions
performed
using unmodified primers and a fluorescence-quenched dual-labeled probe (DLP),
5'-nuclease assay format. The reaction was performed with or without template
(HeLa
cDNA) as indicated. B) Reactions performed using a fluorescence-quenched FQT
For
primer and an unmodified Rev primer in a primer-probe assay format. Reactions
were
performed with or without the addition of RNase H2 as indicated. Cycle number
is
shown on the X-axis and relative fluorescence intensity is shown on the Y-
axis.
[074] Figure 33 shows the sequences of cleavable-blocked primers that are
either
perfect match or contain a mismatch at position +2 relative to the single RNA
base (2
bases 3'- to the ribonucleotide). SMAD7 target sequences at SNP site rs4939827
are
aligned below the primers to indicate how this strategy results in the
presence of a
single mismatch when primers hybridize with one allele vs. a double mismatch
when
hybridize with the second allele. DNA bases are uppercase, RNA bases are
lowercase,
and SpC3 is a Spacer C3 modification.
16
CA 02949315 2016-11-23
[075] Figure 34 is a graph that shows the relative functional activity of
different
oligonucleotide compositions to prime DNA synthesis in a linear primer
extension
reaction.
[076] Figure 35 shows the scheme for performing cycles of DNA sequencing by
ligation using RNase H2 cleavable ligation probes.
[077] Figure 36 shows the scheme for hybridization, ligation, and
subsequent
cleavage by RNase H2 of RNA-containing cleavable ligation probes of a set of
specific
exemplary synthetic sequences.
[078] Figure 37 shows a photograph of a polyacrylamide gel used to separate
products from ligation reactions done using cleavable ligation probes on a
synthetic
template showing that the 9mer probes are efficiently ligated to the acceptor
nucleic
acid (ANA) and that the ligation product is efficiently cleaved by RNase H2,
leaving an
ANA species that is lengthened by one base. The nucleic acids were imaged
using
fluorescent staining and the image was inverted for clarity.
[079] Figure 38 shows the scheme for hybridization and ligation of
RNA-containing cleavable ligation probes containing either three or four 5-
nitroindole
residues.
[080] Figure 39 shows a photograph of a polyacrylamide gel used to separate
products from ligation reactions done using cleavable ligation probes on a
synthetic
template showing that an 8mer probe containing three 5-nitroindole (3x5NI)
bases is
efficiently ligated to an acceptor nucleic acid (ANA) whereas an 8mer probe
containing
four 5-nitroindole (4x5NI) bases is not. The nucleic acids were imaged using
fluorescent staining and the image was inverted for clarity.
10811 Figure 40 shows a photograph of a polyacrylamide gel used to separate
ligation products from reactions done using cleavable ligation probes on a
synthetic
template showing that an 8mer probe containing a single fixed DNA base at the
5'-end,
four random bases, and 3 universal base 5-nitroindolcs can specifically ligate
to the
target as directed by the single fixed DNA base.
17
CA 02949315 2016-11-23
[0821 Figure 41 shows the scheme for a traditional oligonucleotide ligation
assay
(OLA). Panel A shows the three oligonucleotides needed to interrogate a two
allele
target system. Panel B shows the steps involved in making a ligation product.
[0831 Figure 42 shows the scheme for the RNase H2 cleavable oligonucleotide
ligation assay (OLA) of the present invention. Panel A shows the four
oligonucleotides
needed to interrogate a two allele target system. Panel B shows the steps
involved in
making a ligation product using the RNase H2 method. Panel C illustrates how
this
method tests the identity of the base polymorphism twice.
10841 Figure 43 shows alignment of sequences used in the present Example
during
each step of the RNase H2 cleavable probe OLA using fluorescence microbeads
and a
LurninexTM L100 system to detect the ligation products.
[0851 Figure 44 is a chart that shows the resulting fluorescent signal
detected by a
Luminex L100 system to assess identity of the reaction products generated from
the
RNase H2 allelic discrimination OLA shown in Figure 43.
DETAILED DESCRIPTION OF THE INVENTION
10861 The current invention provides novel nucleic acid compounds having a
cleavage domain and a 3' or 5' blocking group. These compounds offer
improvements
to existing methods for nucleic acid amplification, detection, ligation,
sequencing and
synthesis. New assay formats comprising the use of these novel nucleic acid
compounds are also provided.
Definitions
[0871 To aid in understanding the invention, several terms are defined
below.
[088] The terms "nucleic acid" and "oligonucleotide," as used herein, refer
to
polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleoti des
(containing D-ribose), and to any other type of polynucleotidc which is an N
glycoside
of a purine or pyrimidine base. There is no intended distinction in length
between the
terms "nucleic acid", "oligonucleotide" and "polynucleotide", and these terms
will be
used interchangeably. These terms refer only to the primary structure of the
molecule.
Thus, these terms include double- and single-stranded DNA, as well as double-
and
18
CA 02949315 2016-11-23
single-stranded RNA. For use in the present invention, an oligonucleotide also
can
comprise nucleotide analogs in which the base, sugar or phosphate backbone is
modified as well as non-purinc or non-pyrimidine nucleotide analogs.
[0891 Oligonucleotides can be prepared by any suitable method, including
direct
chemical synthesis by a method such as the phosphotriester method of Narang et
al.,
1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al.,
1979,
Meth. Enzymol. 68:109-151; the diethylphosphoramiditc method of Beaucage et
al.,
1981, Tetrahedron Lett. 22:1859-1862; and the solid support method of U.S.
Pat. No.
4,458,066. A review of synthesis methods of
conjugates of oligonucleotides and modified nucleotides is provided in
Goodchild,
1990, Bioconjugate Chemistry 1(3): 165-187.
[090] The term "primer," as used herein, refers to an oligonucleotide
capable of
acting as a point of initiation of DNA synthesis under suitable conditions.
Such
conditions include those in which synthesis of a primer extension product
complementary to a nucleic acid strand is induced in the presence of four
different
nucleoside triphosphates and an agent for extension (e.g., a DNA polymerase or
reverse
transcriptase) in an appropriate buffer and at a suitable temperature. Primer
extension
can also be carried out in the absence of one or more of the nucleotide
triphosphates in
which case an extension product of limited length is produced. As used herein,
the term
"primer" is intended to encompass the oligonucleotides used in ligation-
mediated
reactions, in which one oligonucleotide is "extended" by ligation to a second
oligonucleotide which hybridizes at an adjacent position. Thus, the term
"primer
extension", as used herein, refers to both the polymerization of individual
nucleoside
triphosphates using the primer as a point of initiation of DNA synthesis and
to the
ligation of two oligonucleotides to form an extended product.
[091] A primer is preferably a single-stranded DNA. The appropriate length
of a
primer depends on the intended use of the primer but typically ranges from 6
to 50
nucleotides, preferably from 15-35 nucleotides. Short primer molecules
generally
require cooler temperatures to form sufficiently stable hybrid complexes with
the
template. A primer need not reflect the exact sequence of the template nucleic
acid, but
must be sufficiently complementary to hybridize with the template. The design
of
19
CA 02949315 2016-11-23
suitable primers for the amplification of a given target sequence is well
known in the art
and described in the literature cited herein.
10921 Primers can
incorporate additional features which allow for the detection or
immobilization of the primer but do not alter the basic property of the
primer, that of
acting as a point of initiation of DNA synthesis. For example, primers may
contain an
additional nucleic acid sequence at the 5' end which does not hybridize to the
target
nucleic acid, but which facilitates cloning or detection of the amplified
product. The
region of the primer which is sufficiently complementary to the template to
hybridize is
referred to herein as the hybridizing region.
[0931 The terms "target,
"target sequence", "target region", and "target nucleic
acid," as used herein, are synonymous and refer to a region or sequence of a
nucleic
acid which is to be amplified, sequenced or detected.
[094] The term
"hybridization," as used herein, refers to the formation of a duplex
structure by two single-stranded nucleic acids due to complementary base
pairing.
Hybridization can occur between fully complementary nucleic acid strands or
between
"substantially complementary" nucleic acid strands that contain minor regions
of
mismatch. Conditions under which hybridization of fully complementary nucleic
acid
strands is strongly preferred are referred to as "stringent hybridization
conditions" or
"sequence-specific hybridization conditions". Stable duplexes of
substantially
complementary sequences can be achieved under less stringent hybridization
conditions; the degree of mismatch tolerated can be controlled by suitable
adjustment
of the hybridization conditions. Those skilled in the art of nucleic acid
technology can
determine duplex stability empirically considering a number of variables
including, for
example, the length and base pair composition of the oligonucleotides, ionic
strength,
and incidence of mismatched base pairs, following the guidance provided by the
art
(see, e.g., Sambrook et al., 1989, Molecular Cloning¨A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical
Review in Biochem. and Mol. Biol. 26(3/4):227-259; and Owczarzy et al., 2008,
Biochemistry, 47: 5336-5353).
10951 The term
"amplification reaction" refers to any chemical reaction, including
an enzymatic reaction, which results in increased copies of a template nucleic
acid
CA 02949315 2016-11-23
sequence or results in transcription of a template nucleic acid. Amplification
reactions
include reverse transcription, the polymerase chain reaction (PCR), including
Real
Time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide
to
Methods and Applications (Innis et al., eds, 1990)), and the ligase chain
reaction (LCR)
(see Barany et al., U.S. Pat. No. 5,494,810). Exemplary "amplification
reactions
conditions" or "amplification conditions" typically comprise either two or
three step
cycles. Two step cycles have a high temperature denaturation step followed by
a
hybridization/elongation (or ligation) step. Three step cycles comprise a
denaturation
step followed by a hybridization step followed by a separate elongation or
ligation step.
[096] As used herein, a "polymerase" refers to an enzyme that catalyzes the
polymerization of nucleotides. Generally, the enzyme will initiate synthesis
at the
3'-end of the primer annealed to a nucleic acid template sequence. "DNA
polymerase"
catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases
include, for example, Pyrococcus .furiosus (Pfu) DNA polymerase (Lundberg et
al.,
1991, Gene, 108:1), E. coli DNA polymerase I (Lecomte and Doubleday, 1983,
Nucleic Acids Rcs. 11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J.
Biol.
Chem. 256:3112), Therms thertnophilus (Tth) DNA polymerase (Myers and Gelfand
1991, Biochemistry 30:7661), Bacillus stearothertnophilus DNA polymerase
(Stenesh
and McGowan, 1977, Biochim Biophys Acta 475:32), Thermococcus litoralis (Tli)
DNA polymerase (also referred to as Vent DNA polymerase, Cariello et al.,
1991,
Nucleic Acids Res, 19: 4193), Thermotoga maritima (Tma) DNA polymerase (Diaz
and Sabin , 1998 Braz J. Med. Res, 31:1239), Therms aquaticus (Taq) DNA
polymerase (Chien et al., 1976, J. Bacteoriol, 127: 1550), Pyrococcus
kodakaraensis
KOD DNA polymcrasc (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504),
JDF-3 DNA polymerase (Patent application WO 0132887), and Pyrococcus GB-D
(PGB-D) DNA polymerase (Juncosa-Ginesta et al., 1994, Biotechniques, 16:820).
The
polymerase activity of any of the above enzymes can be determined by means
well
known in the art.
[097] As used herein, a primer is "specific," for a target sequence if,
when used in
an amplification reaction under sufficiently stringent conditions, the primer
hybridizes
primarily to the target nucleic acid. Typically, a primer is specific for a
target sequence
if the primer-target duplex stability is greater than the stability of a
duplex formed
21
CA 02949315 2016-11-23
between the primer and any other sequence found in the sample. One of skill in
the art
will recognize that various factors, such as salt conditions as well as base
composition
of the primer and the location of the mismatches, will affect the specificity
of the primer,
and that routine experimental confirmation of the primer specificity will be
needed in
many cases. Hybridization conditions can be chosen under which the primer can
form
stable duplexes only with a target sequence. Thus, the use of target-specific
primers
under suitably stringent amplification conditions enables the selective
amplification of
those target sequences which contain the target primer binding sites.
10981 The term "non-specific amplification," as used herein, refers to the
amplification of nucleic acid sequences other than the target sequence which
results
from primers hybridizing to sequences other than the target sequence and then
serving
as a substrate for primer extension. The hybridization of a primer to a non-
target
sequence is referred to as "non-specific hybridization" and is apt to occur
especially
during the lower temperature, reduced stringency, pre-amplification
conditions, or in
situations where there is a variant allele in the sample having a very closely
related
sequence to the true target as in the case of a single nucleotide polymorphism
(SNP).
10991 The term "primer dimer," as used herein, refers to a template-
independent
non-specific amplification product, which is believed to result from primer
extensions
wherein another primer serves as a template. Although primer dimers frequently
appear to be a concatamer of two primers, i.e., a dimer, concatamers of more
than two
primers also occur. The term "primer dimer" is used herein generically to
encompass a
template-independent non-specific amplification product.
101001 The term "reaction mixture," as used herein, refers to a solution
containing
reagents necessary to carry out a given reaction. An "amplification reaction
mixture",
which refers to a solution containing reagents necessary to carry out an
amplification
reaction, typically contains oligonucleotide primers and a DNA polymerase or
ligase in
a suitable buffer. A "PCR reaction mixture" typically contains oligonucleotide
primers,
a DNA polymerase (most typically a thermostable DNA polymcrase), dNTP's, and a
divalent metal cation in a suitable buffer. A reaction mixture is referred to
as complete
if it contains all reagents necessary to enable the reaction, and incomplete
if it contains
only a subset of the necessary reagents. It will be understood by one of skill
in the art
that reaction components are routinely stored as separate solutions, each
containing a
22
CA 02949315 2016-11-23
subset of the total components, for reasons of convenience, storage stability,
or to allow
for application-dependent adjustment of the component concentrations, and that
reaction components are combined prior to the reaction to create a complete
reaction
mixture. Furthermore, it will be understood by one of skill in the art that
reaction
components arc packaged separately for commercialization and that useful
commercial
kits may contain any subset of the reaction components which includes the
blocked
primers of the invention.
101011 For the purposes of this invention, the terms "non-activated" or
"inactivated," as used herein, refer to a primer or other oligonucleotide that
is incapable
of participating in a primer extension reaction or a ligation reaction because
either DNA
polymerase or DNA ligase cannot interact with the oligonucleotide for their
intended
purposes. In some embodiments when the oligonucleotide is a primer, the
non-activated state occurs because the primer is blocked at or near the 3'-end
so as to
prevent primer extension. When specific groups are bound at or near the 3'-end
of the
primer, DNA polymerase cannot bind to the primer and extension cannot occur. A
non-activated primer is, however, capable of hybridizing to a substantially
complementary nucleotide sequence.
[0102] For the purposes of this invention, the term "activated," as used
herein,
refers to a primer or other oligonucleotide that is capable of participating
in a reaction
with DNA polymerase or DNA ligase. A primer or other oligonucleotide becomes
activated after it hybridizes to a substantially complementary nucleic acid
sequence and
is cleaved to generate a functional 3'- or 5'-end so that it can interact with
a DNA
polymerase or a DNA ligase. For example, when the oligonucleotide is a primer,
and
the primer is hybridized to a template, a 3'-blocking group can be removed
from the
primer by, for example, a cleaving enzyme such that DNA polymerase can bind to
the
3' end of the primer and promote primer extension.
[0103] The term "cleavage domain" or "cleaving domain," as used herein, are
synonymous and refer to a region located between the 5' and 3' end of a primer
or other
oligonucleotide that is recognized by a cleavage compound, for example a
cleavage
enzyme, that will cleave the primer or other oligonucleotide. For the purposes
of this
invention, the cleavage domain is designed such that the primer or other
oligonucleotide is cleaved only when it is hybridized to a complementary
nucleic acid
23
CA 02949315 2016-11-23
sequence, but will not be cleaved when it is single-stranded. The cleavage
domain or
sequences flanking it may include a moiety that a) prevents or inhibits the
extension or
ligation of a primer or other oligonucleotide by a polymerase or a ligase, b)
enhances
discrimination to detect variant alleles, or c) suppresses undesired cleavage
reactions.
One or more such moieties may be included in the cleavage domain or the
sequences
flanking it.
101041 The term "RNase H cleavage domain," as used herein, is a type of
cleavage
domain that contains one or more ribonucleic acid residue or an alternative
analog
which provides a substrate for an RNase H. An RNase H cleavage domain can be
located anywhere within a primer or oligonucleotide, and is preferably located
at or
near the 3'-end or the 5'-end of the molecule.
101051 An "RNase HI cleavage domain" generally contains at least three
residues.
An "RNase H2 cleavage domain" may contain one RNA residue, a sequence of
contiguously linked RNA residues or RNA residues separated by DNA residues or
other chemical groups. In one embodiment, the RNase H2 cleavage domain is a
2'-fluoronucleoside residue. In a more preferred embodiment the RNase H2
cleavable
domain is two adjacent 2'-fluoro residues.
101061 The terms "cleavage compound," or "cleaving agent" as used herein,
refers
to any compound that can recognize a cleavage domain within a primer or other
oligonucleotide, and selectively cleave the oligonucleotide based on the
presence of the
cleavage domain. The cleavage compounds utilized in the invention selectively
cleave
the primer or other oligonucleotide comprising the cleavage domain only when
it is
hybridized to a substantially complementary nucleic acid sequence, but will
not cleave
the primer or other oligonucleotide when it is single stranded. The cleavage
compound
cleaves the primer or other oligonucleotide within or adjacent to the cleavage
domain.
The term "adjacent," as used herein, means that the cleavage compound cleaves
the
primer or other oligonucleotide at either the 5'-end or the 3' end of the
cleavage domain.
Cleavage reactions preferred in the invention yield a 5'-phosphate group and a
3'-OH
group.
101071 In a preferred embodiment, the cleavage compound is a "cleaving
enzyme."
A cleaving enzyme is a protein or a ribozyme that is capable of recognizing
the cleaving
24
CA 02949315 2016-11-23
domain when a primer or other nucleotide is hybridized to a substantially
complementary nucleic acid sequence, but that will not cleave the
complementary
nucleic acid sequence (i.e., it provides a single strand break in the duplex).
The
cleaving enzyme will also not cleave the primer or other oligonucleotide
comprising
the cleavage domain when it is single stranded. Examples of cleaving enzymes
are
RNase H enzymes and other nicking enzymes.
101081 The term "nicking," as used herein, refers to the cleavage of only
one strand
of the double-stranded portion of a fully or partially double-stranded nucleic
acid. The
position where the nucleic acid is nicked is referred to as the "nicking site"
(NS). A
"nicking agent" (NA) is an agent that nicks a partially or fully double-
stranded nucleic
acid. It may be an enzyme or any other chemical compound or composition. In
certain
embodiments, a nicking agent may recognize a particular nucleotide sequence of
a fully
or partially double-stranded nucleic acid and cleave only one strand of the
fully or
partially double-stranded nucleic acid at a specific position (i.e., the NS)
relative to the
location of the recognition sequence. Such nicking agents (referred to as
"sequence
specific nicking agents") include, but are not limited to, nicking
endonucleases (e.g.,
N .BstNB).
[01091 A "nicking endonuclease" (NE), as used herein, thus refers to an
endonuclease that recognizes a nucleotide sequence of a completely or
partially
double-stranded nucleic acid molecule and cleaves only one strand of the
nucleic acid
molecule at a specific location relative to the recognition sequence. In such
a case the
entire sequence from the recognition site to the point of cleavage constitutes
the
"cleavage domain".
101101 The term "blocking group," as used herein, refers to a chemical
moiety that
is bound to the primer or other oligonucleotide such that an amplification
reaction does
not occur. For example, primer extension and/or DNA ligation does not occur.
Once
the blocking group is removed from the primer or other oligonucleotide, the
oligonucleotide is capable of participating in the assay for which it was
designed (PCR,
ligation, sequencing, etc). Thus, the "blocking group" can be any chemical
moiety that
inhibits recognition by a polymerase or DNA ligase. The blocking group may be
incorporated into the cleavage domain but is generally located on either the
5'- or
3'-side of the cleavage domain. The blocking group can be comprised of more
than one
CA 02949315 2016-11-23
chemical moiety. In the present invention the "blocking group" is typically
removed
after hybridization of the oligonucleotide to its target sequence.
[0111] The term "fluorescent generation probe" refers either to a) an
oligonucleotide having an attached fluorophore and quencher, and optionally a
minor
groove binder or to b) a DNA binding reagent such as SYBR Green dye.
[0112] The terms "fluorescent label" or "fluorophore" refers to compounds
with a
fluorescent emission maximum between about 350 and 900 nm. A wide variety of
fluorophores can be used, including but not limited to: 5-FAM (also called
5-carboxyfluorescein; also called Spiro(isobenzofuran-1(3H),
9'-(9H)xanthene)-5-carboxylic acid,3',6'-dihydroxy-3-oxo-6-
carboxyfluorescein);
5-flexachloro-Fluorescein;
([4,7,2',4',5',T-hexachloro-(3',6'-dipivaloyl-fluoresceiny1)-6-carboxylic
acid]);
6-Hexachloro-Fluorescein;
([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceiny1)-5-carboxylic
acid]);
5-Tetrachloro-Fluorescein;
([4,7,2',7'-tetra-chloro-(3',6'-dipivaloylfluoresceiny1)-5-carboxylic acid]);
6-Tetrachloro-Fluorescein;
([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceiny1)-6-carboxylic acid]); 5-
TAMRA
(5-carboxytetramethylrhodamine); Xanthylium,
9-(2,4-dicarboxypheny1)-3,6-bis(dimethyl-amino); 6-TAMRA
(6-carboxytetramethylrhodamine); 9-(2,5-dicarboxypheny1)-3,6-
bis(dimethylamino);
EDANS (5-((2-aminoethyl)amino)naphthalene-1 -sulfonic acid); 1,5-IAEDANS
(5((((2-iodoacetypamino)ethypamino)naphthalenc-1-sulfonic acid); Cy5
(Indodicarbocyanine-5); Cy3 (Indo-dicarbocyanine-3); and BODIPY FL
(2,6-dibromo-4,4-difluoro-5,7-dimethy1-4-bora-3a,4a-diaza-s-indacene-3-
proprionic
acid); Quasar' -670 dye (Bioscarch Technologies); Cal Fluor Orange dye
(Biosearch
Technologies); Rox dyes; Max dyes (Integrated DNA Technologies), as well as
suitable derivatives thereof.
[0113] As used herein, the term "quencher" refers to a molecule or part of
a
compound, which is capable of reducing the emission from a fluorescent donor
when
attached to or in proximity to the donor. Quenching may occur by any of
several
mechanisms including fluorescence resonance energy transfer, photo-induced
electron
26
CA 02949315 2016-11-23
transfer, paramagnetic enhancement of intersystem crossing, Dexter exchange
coupling,
and exciton coupling such as the formation of dark complexes. Fluorescence is
quenched" when the fluorescence emitted by the fluorophore is reduced as
compared
with the fluorescence in the absence of the quencher by at least 10%, for
example, 15%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9% or more. A
number of commercially available quenchers are known in the art, and include
but are
not limited to DABCYL, Black HoleTm Quenchers (BHQ-1, BHQ-2, and BHQ-3),
Iowa Blade' FQ and Iowa Blade RQ. These are so-called dark quenchers. They
have
no native fluorescence, virtually eliminating background problems seen with
other
quenchers such as TAMRA which is intrinsically fluorescent.
[0114] The term "ligation" as used herein refers to the covalent joining of
two
polynucleotide ends. In various embodiments, ligation involves the covalent
joining of
a 3' end of a first polynucleotide (the acceptor) to a 5' end of a second
polynucleotide
(the donor). Ligation results in a phosphodiester bond being formed between
the
polynucleotide ends. In various embodiments, ligation may be mediated by any
enzyme, chemical, or process that results in a covalent joining of the
polynucleotide
ends. In certain embodiments, ligation is mediated by a ligase enzyme.
[0115] As used herein, "ligase" refers to an enzyme that is capable of
covalently
linking the 3' hydroxyl group of one polynucleotide to the 5' phosphate group
of a
second polynucleotide. Examples of ligases include E. coil DNA ligase, T4 DNA
ligase, etc.
[0116] The ligation reaction can be employed in DNA amplification methods
such
as the "ligase chain reaction" (LCR), also referred to as the "ligase
amplification
reaction" (LAR), see Barany, Proc. Natl. Acad. Sci., 88:189 (1991); and Wu and
Wallace, Gcnomics 4:560 (1989). In LCR, four
oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to
one strand
of the target DNA, and a complementary set of adjacent oligonucleotides, that
hybridize to the opposite strand are mixed and DNA ligase is added to the
mixture. In
the presence of the target sequence, DNA ligase will covalently link each set
of
hybridized molecules. Importantly, in LCR, two oligonucleotides are ligated
together
only when they base-pair with sequences without gaps. Repeated cycles of
denaturation, hybridization and ligation amplify a short segment of DNA. A
mismatch
27
CA 02949315 2016-11-23
at the junction between adjacent oligonucleotides inhibits ligation. As in
other
oligonucicotide ligation assays this property allows LCR to be used to
distinguish
between variant alleles such as SNPs. LCR has also been used in combination
with
PCR to achieve enhanced detection of single-base changes, see Segev, PCT
Public. No.
W09001069 (1990).
Novel oligonucleotides and compounds of the present invention.
01171 In one embodiment, the novel oligonucleotides of the present
invention are
primers for DNA replication, as for example in PCR, DNA sequencing and
polynomial
amplification, to name a few such applications. In this embodiment, the
primers have
an inactive configuration wherein DNA replication (i.e., primer extension) is
blocked,
and an activated configuration wherein DNA replication proceeds. The inactive
configuration of the primer is present when the primer is either single-
stranded, or the
primer is hybridized to the DNA sequence of interest and primer extension
remains
blocked by a chemical moiety that is linked at or near to the 3' end of the
primer. The
activated configuration of the primer is present when the primer is hybridized
to a
nucleic acid sequence of interest and subsequently acted upon by RNase H or
other
cleaving agent to remove the blocking group and allow for an enzyme (e.g., a
DNA
polymcrasc) to catalyze primer extension.
101181 A number of blocking groups arc known in the art that can be placed
at or
near the 3' end of the oligonucleotide (e.g., a primer) to prevent extension.
A primer or
other oligonucicotide may be modified at the 3'-terminal nucleotide to prevent
or
inhibit initiation of DNA synthesis by, for example, the addition of a 3'
deoxyribonucleotide residue (e.g., cordycepin), a 2',3 ' -
dideoxyribonucleotide residue,
non-nucleotide linkages or alkane-diol modifications (U.S. Pat. No.
5,554,516).
Alkane diol modifications which can be used to inhibit or block primer
extension have
also been described by Wilk et al., (1990, Nucleic Acids Res., 18 (8):2065),
and by
Arnold et al., (U.S. Pat. No. 6,031,091). Additional examples of suitable
blocking
groups include 3' hydroxyl substitutions (e.g., 3'-phosphatc, 3'-triphosphatc
or
3'-phosphate diesters with alcohols such as 3- hydroxypropyl), a 2'3'-cyclic
phosphate,
2' hydroxyl substitutions of a terminal RNA base (e.g., phosphate or
sterically bulky
groups such as triisopropyl silyl (TIPS) or tert-butyl dimethyl silyl
(TBDMS)).
2'-alkyl silyl groups such as TIPS and TBDMS substituted at the 3'-end of an
28
CA 02949315 2016-11-23
oligonucleotide are described by Laikhter et al., U.S. Pat. App. Serial No.
11/686,894.
Bulky substituents can also be incorporated
on the base of the 3'-terminal residue of the oligonucleotide to block primer
extension.
101191 Blocking groups to inhibit primer extension can also be located
upstream,
that is 5', from the 3'-terminal residue. Sterically bulky substituents which
interfere
with binding by the polymerase can be incorporated onto the base, sugar or
phosphate
group of residues upstream from the 3'-terminus. Such substituents include
bulky alkyl
groups like t-butyl, triisopropyl and polyaromatic compounds including
fluorophores
and quenchers, and can be placed from one to about 10 residues from the 3'-
terminus.
Alternatively abasic residues such as a C3 spacer may be incorporated in these
locations to block primer extension. In one such embodiment two adjacent C3
spacers
have been employed (see Examples 27 and 28).
101201 In the case of PCR, blocking moieties upstream of the 3'-terminal
residue
can serve two functions: 1) to inhibit primer extension, and 2) to block the
primer from
serving as a template for DNA synthesis when the extension product is copied
by
synthesis from the reverse primer. The latter is sufficient to block PCR even
if primer
extension can occur. C3 spacers placed upstream of the 3'-terminal residue can
function in this manner (see Examples 26 and 27).
[01211 A modification used as a blocking group may also be located within a
region
3' to the priming sequence that is non-complementary to the target nucleic
acid
sequence.
[01221 The oligonucleotide further comprises a cleavage domain located
upstream
of the blocking group used to inhibit primer extension. An RNase H cleavage
domain
is preferred. An RNase H2 cleavage domain comprising a single RNA residue or
replacement of the RNA base with one or more alternative nucleosides is most
preferred.
[01231 In one embodiment, RNasc H2 can be used to cleave duplexes
containing a
single 2'-fluoro residue. Cleavage occurs on the 5' side of the 2'-fluoro
residue. In a
preferred embodiment, an RNase H2 cleavage domain comprising two adjacent
2'-fluoro residues is employed (see Example 6). The activity is enhanced when
two
29
CA 02949315 2016-11-23
consecutive 2'-fluoro modifications are present. In this embodiment cleavage
occurs
preferentially between the 2'-fluoro residues. Unlike oligonucleotides
containing
unmodified RNA residues, oligonucleotides with 2'-fluoro groups are not
cleaved by
single-stranded ribonucleases and are resistant to water catalyzed cleavage
and
completely stable at high temperatures. Enhanced cleavage has also been found
when a
2'-fluoro modified RNA residue is used with a 2' LNA modified RNA residue.
2'-fluoro-containing oligonucleotides have been found to be further
advantageous in
certain applications compared to RNA-containing oligonucleotides in offering
greater
discrimination with respect to mismatches between the oligonucleotide and the
target
sequence.
[0124] Alternatives to an RNA residue that can be used in the present
invention
wherein cleavage is mediated by an RNase H enzyme include but are not limited
to
2'-0-alkyl RNA nucleosides, preferably 2'-0-methyl RNA nucleosides,
2'-fluoronucleosides, locked nucleic acids (LNA), 2'-ENA residues (ethylene
nucleic
acids), 2'-alkyl nucleosides, 2'-aminonucleosides and 2'-thionucleosides. The
RNase
El cleavage domain may include one or more of these modified residues alone or
in
combination with RNA bases. DNA bases and abasic residues such as a C3 spacer
may
also be included to provide greater performance.
101251 if the cleaving agent is an RNase HI enzyme a continuouse sequence
of at
least three RNA residues is preferred. A continuous sequence of four RNA
residues
generally leads to maximal activity. If the cleaving agent is an RNase H2
enzyme a
single RNA residue or 2 adjacent 2'-fluoro residues are preferred.
[0126] One objective of incorporating modified residues within an RNase H
cleavage domain is to suppress background cleavage of a primer or probe due to
water
catalyzed hydrolysis or cleavage by single stranded ribonucleases. Replacement
of the
2'-hydroxyl group with a substituent that cannot attack the adjacent phosphate
group of
an RNA residue can accomplish this goal. Examples of this approach include the
use of
the 2'-substituted nucleosides listed above, such as 2'-fluoro and 2'-0-methyl
nucleosides. This is particularly advantageous when cleavage is mediated by
RNase
H2 and there is a single RNA residue within the cleavage domain. As shown in
Figure
3, in this case cleavage by single stranded ribonucleases or water catalyzed
hydrolysis
occurs at a different position than cleavage by RNase H2.
CA 02949315 2016-11-23
101271 Other examples of modifications that can be used to suppress
cleavage by
single stranded ribonucleases and water catalyzed hydrolysis at RNA residues
include
substitution of the 5' oxygen atom of the adjacent residue (3'- to the RNA
base) with an
amino group, thiol group, or a methylene group (a phosphonate linkage).
Alternatively
one or both of the hydrogen atoms on the 5' carbon of the adjacent residue can
be
replaced with bulkier substituents such as methyl groups to inhibit background
cleavage of a ribonucleotide residue. In another such embodiment, the
phosphate group
at the 3'-side of an RNA residue can be replaced with a phosphorothioate,
phosphorodithioates or boronate linkage. In the case of a phosphorothioate the
S
stereoisomer is preferred. Combinations of these various modifications may
also be
employed.
[0128] It should be noted that background cleavage at RNA residues by
single
stranded ribonucleascs or water catalyzed hydrolysis leads to a blocked 3'-end
(see
Figure 3) that cannot serve as a primer for DNA synthesis. This mitigates the
occurrence of false positive results even if such cleavage does occur.
101291 The cleavage domain may include the blocking group provided that
cleavage occurs on the 5 '-side of the blocking group and generates a free 3 '-
OH.
Generally however the cleavage domain and the blocking group are separated by
one to
about 15 bases. After cleavage takes place the portion of the primer 3' from
the
cleavage site containing the blocking group dissociates from the template and
a
functional 3'-hydroxyl group is exposed, capable of being acted on by a
polymerase
enzyme. The optimal distance between the cleavage site and the blocking group
will
depend on the cleaving agent and the nature of the blocking group. When
cleavage of
the oligonucleotide is mediated by RNase H2 at a single RNA residue a distance
of 3 to
about 8 bases between the cleavage site and the blocking group is preferred.
If the
blocking group is sterically small, for example a phosphodiester at the 3'
terminal
nucleotide as in the following structure
31
CA 02949315 2016-11-23
'V3-7
.04.0
6
(C1-1213-0 H
a cleavage site 5 bases from the 3'-end is generally optimal. If the blocking
group is
larger it is advantageous to position the cleavage site further from it.
101301 In a preferred embodiment, a thermophilic RNase H2 enzyme is
utilized to
cleave the oligonucleotide. In yet a more preferred embodiment, a thermophilic
RNase
H2 enzyme is used which is less active at room temperature than at elevated
temperatures. This allows a hot-start type of reaction to be achieved in PCR
and other
primer extension assays using the blocked primers of the present invention
without
actually requiring a hot start, i.e., reversibly inactivated, DNA polymerase.
Standard
less expensive DNA polymerase polymerases such as Taq polymerase can be used
instead of the much more expensive hot start versions of the enzyme. Moreover,
for
different applications alternative DNA polymerases may be preferred. Utilizing
RNase
H as the hot start component of the assay obviates the need to develop a new
reversibly
inactivated analog of each different DNA polymerase.
101311 Hot start properties of the enzyme may be intrinsic to the protein
as in the
case of Pyrocoecus abysii RNase H2 (see Example 4). Alternatively the enzyme
may
be reversibly inactivated by chemical modification using, for example, maleic
acid
anhydride analogs such as citroconic anhydride. These compounds react with
amino
groups of the protein and at high temperature are released restoring activity.
In yet
another embodiment antibodies against an RNase H which block the enzyme may be
employed which are denatured at elevated temperatures.
101321 In yet another embodiment, the oligonucleotide of the present
invention has
a cleavage domain that is recognized and cleaved by a sequence specific
nicking agent,
e.g., a nicking enzyme. The nicking agent also can be designed to cleave an
oligonucleotide (e.g., a primer) at a modified nucleic acid or grouping of
modified
nucleic acids. In this embodiment, the oligonucleotide is designed to be
recognized by
32
CA 02949315 2016-11-23
a nicking agent upon hybridization with the target nucleic acid, and the
nicking of the
oligonucleotide/target duplex can be used to remove a blocking group and allow
for
oligonucleotide extension. The nicking site (NS) is preferably located at or
near the
3'-end of the oligonucleotide, specifically, one to about 15 bases from the 3'-
end of the
oligonucleotide.
101331 Exemplary nicking agents include, without limitation, single strand
nicking
restriction endonucleases that recognize a specific sequence such as N.BstNBI;
or
repair enzymes such as Mut H, MutY (in combination with an AP endonuclease),
or
uracil-N-glycosylase (in combination with an AP Lyase and AP endonucleases);
and
the genell protein of bacteriophage fl.
101341 The blocked primers of the present invention minimize non-specific
reactions by requiring hybridization to the target followed by cleavage before
primer
extension. If a primer hybridizes incorrectly to a related sequence, cleavage
of the
primer is inhibited especially when there is a mismatch that lies at or near
the cleavage
site. This reduces the frequency of false priming at such locations and
thereby
increases the specificity of the reaction. It should be noted that with
Pyrococcus abysii
Type II RNase H and other RNase H enzymes used in the present invention some
cleavage does occur even when there is a mismatch at the cleavage site.
Reaction
conditions, particularly the concentration of RNase H and the time allowed for
hybridization and extension in each cycle, can be optimized to maximize the
difference
in cleavage efficiencies between the primer hybridized to its true target and
when there
is a mismatch. This allows the methods of the present invention to be used
very
effectively to distinguish between variant alleles, including SNPs (see
Examples 12-14,
22-25).
101351 As noted above, background cleavage of the primer does not lead to
false-positive priming when RNA residues are incorporated into the
oligonucleotide,
because the 2',3'-cyclic phosphate (or 2' or 3'-phosphate) formed at the 3'
end of the
cleaved primer blocks primer extension. A freely accessible 3' OH group is
needed to
form a substrate for DNA polymerase. The formation of primer-dimers, a common
side
reaction occurring in PCR, can also be inhibited using the 3' blocked primers
of the
present invention. This allows for a greater degree of multiplexing in PCR
(e.g.,
33
CA 02949315 2016-11-23
detecting multiple target sequences in the case of a DNA
detection/amplification
assay).
[0136] Without being bound by any theory, it has been observed that
atypical
cleavage can occur at a low frequency 3' to an RNA residue when there is a
mismatch,
presumably catalyzed by RNase H2, to generate a free 3'-OH and lead to primer
extension. This can result in a decrease in the specificity of the reaction.
To mitigate
this effect nuclease resistant residues can be incorporated into the primer 3'
to the RNA
residue (see Example 22, 25 and 28). Such groups include but are not limited
to one or
more phosphorothioates, phosphorodithioates, methyl phosphonates and abasic
residues such as a C3 spacer.
[0137] Other substitutions both 5' and 3' to the RNA residue can also be
utilized to
enhance the discrimination and detection of variant alleles in the methods of
the present
invention. Such substitutions include but are not limited to 2'-0-methyl RNA
and
secondary mismatches (see Example 23).
[0138] The nature of the blocking group which prevents primer extension is
not
critical. Tt can be placed at the 3'-terminal residue or upstream from it.
Labeling
groups can be incorporated within the blocking group or attached at other
positions on
the 3'-segment of the oligonucleotide primer which dissociates from the
template after
cleavage occurs. Such labeling groups include, but are not limited to,
fluorophorcs,
quenchers, biotin, haptens such as digoxigcnin, proteins including enzymes and
antibodies, mass tags which alter the mass of the cleavage fragment for
detection by
mass spectrometry, and radiolabels such as 14C, 3H, 35S, 32P and 33P. These
labeling
groups can also be attached to the primer 5' to the cleavage site, in which
case they will
be incorporated within the extension product.
[0139] In one embodiment, the blocking group at or near the 3'-end of the
oligonucleotide can be a fluorescent moiety. In this case, release of the
fluorescent
molecule can be used to monitor the progress of the primer extension reaction.
This is
facilitated if the oligonucleotide also contains a quencher moiety on the 5'-
side of the
cleavage site. Cleavage of the oligonucleotide during the reaction separates
the
fluorophore from the quencher and leads to an increase in fluorescence. If the
quencher
34
CA 02949315 2016-11-23
is itself a fluorophore, such as Tamra, a decrease in its fluorescence may
also be
observed.
101401 In yet a further embodiment, the oligonucleotide is labeled with a
fluorescent molecule on the 5 '-side of the cleavage domain, and the blocking
group
located at or near the 3'-end of the molecule is a quencher such as Iowa Black
, Black
Holemi, or Tamra to name a few. Again, cleavage of the quencher from the
oligonucleotide (e.g., a primer) leads to an increase in fluorescence which
can be used
to monitor the progress of the oligonucleotide extension reaction. Moreover,
in this
case, the primer extension product is fluorescently labeled.
101411 In yet a further embodiment, the blocked primers of the present
invention
are used for nucleic acid sequencing. As in the case of DNA amplification
reactions,
the specificity of primer extension for DNA sequencing is also increased when
using
the oligonucleotides of the present invention. In one sequencing embodiment,
2',3'
dideoxynucleotide triphosphates that are fluorescently labeled and used as
chain
terminators and the nested fragments produced in the reaction are separated by
electrophoresis, preferably capillary electrophoresis.
10142] In yet another embodiment, an oligonucleotide primer of the present
invention is labeled with a fluorescent group and the 3' dideoxynucleotide
triphosphatc
chain terminators arc unlabeled. In this embodiment, the blocking group can be
a
quencher, in which case background fluorescence is reduced because the primer
itself is
not fluorescent. Only the extension products are fluorescent.
101431 Another aspect of the invention includes the incorporation of
alternative
divalent cations such as Mn2 Ni2 or Co2 with or without Mg2' , into the assay
buffer.
In certain embodiments of the invention, when such alternative divalent
cations are
present, the effectiveness of the particular assay is increased due to
enhanced cleavage
by RNase H2. In one embodiment, when two adjacent 2'-fluoronucleoside residues
constitute the RNase H2 cleavable domain, 0.3-1 mM MnC12 with 2-4 mM MgC12
gave
optimal performance in the assay (see Example 3).
CA 02949315 2016-11-23
The Methods of the Present Invention
[0144] The primers, probes and other novel oligonucleotides described
herein can
be utilized in a number of biological assays. Although the following list is
not
comprehensive, the majority of the methods of the present invention fall into
six
general categories: (1) primer extension assays (including PCR, DNA sequencing
and
polynomial amplification), (2) oligonucleotide ligation assays (OLA), (3)
cycling
probe reactions, (4) sequencing by ligation, (5) sequencing by generation of
end-labeled fragments using RNase H enzymes, and (6) synthesis by ligation.
[0145] The primers, probes and other novel oligonucleotides described
herein can
be utilized in a number of primer extension assays.
Primer Extension Assays
101461 In one embodiment of the present invention, a method of amplifying a
target
DNA sequence of interest is provided. The method comprises the steps of:
(a) providing a reaction mixture comprising a primer having a cleavage
domain and
a blocking group linked at or near to the 3' end of the primer which prevents
primer
extension, a sample nucleic acid having the target DNA sequence of interest, a
cleaving
enzyme and a polymerase;
(b) hybridizing the primer to the target DNA sequence to form a double-
stranded
substrate;
(c) cleaving the hybridized primer with the cleaving enzyme at a point
within or
adjacent to the cleavage domain to remove the blocking group from the primer;
and
(d) extending the primer with the polymerase.
PCR in General
[0147] When used in PCR, a 3'-blocked primer containing a cleavage domain
first
hybridizes to the target sequence. In this embodiment, the primer cannot
extend until
cleavage of the 3' blocking group occurs after hybridization to the
complementary
DNA sequence. For example, when an RNase H cleavage domain is present in the
primer, an RN ase H enzyme will recognize the double-stranded substrate formed
by the
primer and target and cleave the primer within or adjacent to the cleavage
domain. The
primer can then extend and amplification of the target can then occur. Because
the
36
CA 02949315 2016-11-23
primer needs to be recognized and cleaved by RNase H before extension, non-
specific
amplification is reduced.
[0148] In conventional PCR, a "hot start" polymerase is often used to
reduce
primer dimers and decrease non-specific amplification. Blocked primers of the
present
invention requiring cleavage by RNase H can confer the same advantage. A
thermophilic RNasc H enzyme with little or no activity at lower temperatures
is
preferred. Activation of the primers occurs only after hybridization to the
target
sequence and cleavage at elevated temperatures. Advantages of this approach
compared to the use of a hot start reversibly inactivated DNA polymerase have
been
described above. Of course a hot start RNase H enzyme and a hot start DNA
polymerase can be used in conjunction, if desired.
[0149] Three types of hot start RNase H enzymes are described here (see
Tables 1,
2, and 3): 1) a thermostable RNase H enzyme that has intrinsically little or
no activity at
reduced temperatures as in the case of Pyrococcus abysii RNase H2; 2) a
thermostable
RNase H reversibly inactivated by chemical modification; and 3) a thermostable
RNase
H reversibly inactivated by a blocking antibody. In addition, through means
well-known in the art, such as random mutagenesis, mutant versions of RNase H
can be
synthesized that can further improve the traits of RNase H that are desirable
in the
assays of the present invention. Alternatively, mutant strains of other
enzymes that
share the characteristics desirable for the present invention could be used.
[0150[ In one embodiment, the cleavage domain within the primer is
cleavable by
RNase H. In yet a further embodiment, the RNase H cleavage domain consists of
a
single RNA residue and cleavage of the primer is mediated by a Type II RNase H
enzyme, preferably by a thermophilic Type II RNase H enzyme, and even more
preferably a thermophilic Type II RNase H enzyme which is less active at room
temperature than at elevated temperatures. In yet a further embodiment, the
RNase H2
cleavage domain consists of two adjacent 2'-fluoro nucleoside residues. In yet
a more
preferred embodiment of the present invention in which the cleavage domain
consists
of two adjacent 2'-fluoro nucleoside residues, the PCR is carried out in
buffers
containing alternative divalent cations, including but not limited to, Mn2 ,
Ni2 or Co2
in addition to Mg2 . In an additional embodiment, the novel 3'-blocked primers
of the
present invention comprising a cleavage domain can be utilized in a variation
of hot
37
CA 02949315 2016-11-23
start PCR in which a thermophilic nicking enzyme is used and the cleavage
domain is a
nicking site.
[0151] Alternatively, a cleavage enzyme that lacks hot start
characteristics can be
used in the present invention with traditional hot-start methods such as
adding the
enzyme at an elevated temperature, encasing a necessary reagent or enzyme in
wax, or
with a hot start reversibly inactivated DNA polymerase.
[0152] The increased specificity of the present invention, when used in
amplification reactions, enables real-time PCR applications to achieve more
specific
results, as compared to conventional real-time PCR with standard DNA primers.
For
example, double-stranded DNA-binding dye assays, such as SYBR Green assays,
have a disadvantage in that a signal is produced once the dye binds to any
double-stranded product produced by PCR (e.g., a primer dimer) and can thereby
give
rise to a false positive result. But when a primer of the current invention is
used,
non-specific amplification and primer-dimer formation is reduced, and the
intensity of
the signal of the double-stranded DNA-binding dye will reflect amplification
only of
the desired target (see Example 17).
[0153] The reagent concentrations and reaction conditions of the assay can
be
varied to maximize its utility. The relative efficiency of PCR using the
blocked primers
of the present invention relates to the concentration of the unblocking enzyme
and the
dwell time at the anneal/extend reaction temperature (where unblocking
proceeds).
With low amounts of enzyme and short dwell times, cleavage can be incomplete
and
the reactions with blocked primers have lower efficiency than those with
unblocked
primers. As either enzyme concentration or dwell time increases, the reaction
efficiency with blocked primers increases and becomes identical to unblocked
primers.
The use of even more enzyme or longer dwell times can decrease the specificity
of the
assay and lessen the ability of the system to discriminate mismatches at the
cleavage
site or within the surrounding sequence (see Example 4). This results because
there is
an increase in the efficiency of cleavage of the primer when it is hybridized
to a
mismatch sequence. Cleavage at the true target site cannot be further
increased because
it is already at 100% each cycle. Thus the assay can be tuned for SNP assays
requiring
higher specificity, or for quantitation of expression levels of mRNA requiring
less
specificity.
38
CA 02949315 2016-11-23
101541 In another embodiment, a primer pair having one blocked primer and
one
unblocked primer, can be used. In another embodiment, an enzyme can be
selected that
has less sequence specificity and can cleave various sequences. In yet another
embodiment, an additional mismatch flanking the cleavage site can be added to
increase the ability to discriminate variant alleles. Modified bases such as
2'-0-methyl
nucleosides can also be introduced into the primer on either side of the
cleavage site to
increase specificity (see Example 23).
101551 The reactions of the various assays described herein can be
monitored using
fluorescent detection, detection by mass tags, enzymatic detection, and via
labeling the
probe or primer with a variety of other groups including biotin, haptens,
radionucleotides and antibodies to name a few. In one embodiment, the progress
of
PCR using the modified primers of the present invention is monitored in real
time using
a dye intercelating assay with, for example, SYBR Green. In yet a further
embodiment, the progress of PCR using the modified primers of the present
invention
is monitored using a probe labeled with a fluorophore and a quencher such as a
molecular beacon or, as in the 5 'nuclease assay where cleavage of the probe
occurs.
Alternatively, a dual labeled probe which is cleavable by RNase H2 may be
employed.
In the latter case, cleavage of both the hybridized primers and the probe can
be
mediated by the same enzyme. The RNase H cleavage domain within the probe may
comprise only RNA residues. In general, all of the combinations of residues
useful in
the cleavage domain of the blocked primers of the present invention can be
used as the
cleavage domain within the probe. In particular, when RNase H2 is used as the
cleavage enzyme, a single RNA residue or two adjacent 2'-F residues are
preferred as
the cleavage domain within the probe. Such a modified oligonucleotide probe is
particularly useful in real-time PCR and can be employed with standard DNA
primers
or with the blocked primers of the present invention. In such real-time PCR
assays,
thermophilic versions of RNase H2 are preferred, especially thermophilic RNase
H2
enzymes having lower activity at reduced temperatures. In the examples
provided
herein, a number of thermophilic RNase H2 enzymes have been isolated and have
shown to be stable under thermocycling conditions and useful in PCR. When used
with
the blocked primers of the present invention, the need for a specific hot-
start DNA
polymerase can be eliminated. This results in a significant decrease in assay
cost.
39
CA 02949315 2016-11-23
101561 In another embodiment, the blocked primers of the present invention
can be
used in the primer-probe assay format for PCR described in U.S. Patent App.
2009/0068643. In this case, the primer also contains a label domain on the 5'
end of the
oligonucleotide which may or may not be complementary to the target nucleic
acid.
The product generated by extension of the primer serves as a template for
synthesis by
the reverse primer in the next cycle of PCR. This converts the label domain
into a
double stranded structure. In one such embodiment a fluorophore and a quencher
are
attached to the label domain and the reaction is monitored by an increase in
fluprescence resulting from an increase in the distance between the
fluorophore and
quencher in the double stranded form compared to the single stranded state. In
yet
another such embodiment the label domain contains a cleavage domain located
between the fluorophore and quencher. Cleavage occurs only when the cleavage
domain is double stranded. Again the reaction is monitored by an increase in
fluorescence. In this instance the cleaving agent may be one that cleaves both
strands,
the primer and its complement, such as a restriction enzyme. Alternativley the
cleaving
agent may be a nicking agent that cleaves only the primer, preferably an RNase
H
enzyme, and even more preferably a thermostable RNase H2 enzyme. There are two
cleavage domains within the primer in this assay format: one 5' of the
blocking group at
which cleavage occurs to activate the primer and allow extension and the
second within
the label domain. Cleavage at both sites can be mediated by the same cleaving
agent.
The label domain may also contain other labeling groups including but not
limited to
biotin, haptens and enzymes to name a few. Alternatively the 5' fragment
released by
cleavage within the label domain may serve as a mass tag for detection by mass
spectrometry.
(01571 In yet another embodiment, the blocked primers of the present
invention can
be used in the template-probe assay format for PCR described in U.S. Patent
App.
2009/0068643.
101581 In another embodiment of the invention, RNase H2 cleavable blocked
oligonucleotides are used to detect 5-methylcytosine residues by PCR analysis
of
sodium bisulfite treated nucleic acids, including but not limited to DNA and
RNA.
Previous work has established that treatment of nucleic acid template with
bisulfite will
rapidly dcaminate cytosines that arc not methylated on the 5' carbon of the
base. This
CA 02949315 2016-11-23
deamination reaction converts the unmethylated cytosines into uracil,
resulting in a
functional C->T transition mutation in the nucleic acid sequence. It is also
known that
5-methylcytosine is highly resistant to this deamination, resulting in
preservation of the
5-methylcytosine nucleotide as a cytosine, rather than conversion to a
thymine.
Numerous methods have been employed to detect 5' cytosine methylation
modifications following the bisulfite conversion technique. Examples include,
but are
not limited to, standard mismatch-specific quantitative and non-quantitative
PCR
methods, as well as subcloning and sequencing of the generated sodium
bisulfite
reaction products.
[0159] In the present invention, the template is bisulfite treated by
methods that are
well known to those in the art. If the starting template was RNA, a
complementary
cDNA strand is generated by any well known reverse transcription method.
Blocked
cleavable oligonucleotides that will either match or discriminate against the
target
template cytosines (now converted to uracils) or 5-methylcytosines are added
to a PCR
reaction containing the RNasc H2 enzyme and the bisulfite treated template.
Amplification of the mismatched (converted cytosinc>uracil or unconverted
5-methylcytosine>5-methylcytosine) base containing template is highly reduced
relative to the matched base template due to the mismatch discrimination of
RNase H2
cleavage reaction. Incomplete bisulfite conversion of cytosines to uracils, a
consistent
concern with the sodium bisulfite conversion technique, can be detected by the
designing blocked cleavable oligonucleotides that target known non-5'-
methylated
cytosines in the bisulfite converted template. PCR amplification of
unconverted
cytosines with these primers should display greater discrimination relative to
standard
unblocked primers. The present invention is expected to significantly increase
the
discrimination of the methylated and unmethylated cytosines.
Allele Specific PCR
[0160] The blocked primers of the present invention can also be used in
allele-specific PCR (AS-PCR). In general, AS-PCR is used to detect variant
alleles of a
gene, especially single base mutations such as SNPs (see for example U.S.
Patent No.
5,496,699). SNP locations in the genome, as well as sequences of mutated
oncogenes,
are known in the art and PCR primers can be designed to overlap with these
regions.
41
CA 02949315 2016-11-23
[0161] Detection of single base mismatches is a critical tool in diagnosing
and
correlating certain diseases to a particular gene sequence or mutation.
Although
AS-PCR has been known in the biological arts for more than a decade (Bottema
et al.,
1993, Methods Enzymol., 218, pp. 388-402), tools are still needed to more
accurately
discriminate between particular mismatches and fully complementary sequences.
The
present invention addresses this need.
[0162] In AS-PCR a primer is utilized which overlaps the variant locus.
Generally
the primer is designed such that the 3'-terminal nucleotide is positioned over
the
mutation site. Alternatively, the mutation site is sometimes located over one
or two
bases from the 3'-end. If there is a mismatch at or near the 3'-end, primer
extension and
hence PCR are inhibited. The difference between the efficiency of
amplification when
there is an exact match with the primers versus an allelic variant where there
is one or
more mismatches can in some cases be measured by end point PCR in which case
the
final amplification products are analyzed by, for example, gel
electrophoresis. More
commonly real time PCR is used to determine the efficiency of amplification. A
fluorescence based method of detection of the amplicon in real time such as a
DNA dye
binding assay or a dual labeled probe assay is most often used. The PCR cycle
where
fluorescence is first detectable above background levels (the Cp, or crossing
point)
provides a measure of amplification efficiency. If there is a mismatch between
the
primer and the target DNA, amplification efficiency is reduced and the Cp is
delayed.
Generally an increase in Cp of 4 to 5 cycles is sufficient for discrimination
of SNPs.
[0163] In one AS-PCR embodiment of the present invention, the primer
contains a
single RNA residue, and the mismatch can be aligned directly over the RNA
residue of
the primer. The difference in crossing point (Cp) values between a perfect
match and a
mismatch, correlating to a cleavage differential, is readily apparent (see
Example 13).
In some instances, aligning the mismatch one base to either the 5' side or the
3' side of
the RNA residue increases the difference in Cp values. When the mismatch is
located
on the 5' side of the RNA residue, the subsequent RNase H2 cleavage would
leave the
mismatch as the last base of the 3' end of the cleaved primer. Surprisingly,
having the
mismatch directly on top of the RNA residue is more effective in most cases
than
locating the mismatch to the 5' side of the RNA residue.
42
CA 02949315 2016-11-23
[0164] In another embodiment, the primer contains multiple RNA residues or
two
adjacent 2'-fluoro residues and detection of the mismatch follows the same
principles
as with a primer containing one RNA residue; the mismatch preferably is
located near
or on top of the expected point of cleavage.
[0165] In another embodiment, a second mismatch is used to increase the
sensitivity of the assay. In yet a further embodiment, the second mismatch is
placed to
the 3' side of the mismatch directly over the SNP site. In yet a further
embodiment, the
second mismatch is placed one or two bases from the mismatch directly over the
SNP
site (see Example 23).
101661 In yet another embodiment, modified residues are incorporated into
the
primer on the 5'- or 3'-side of the base located over the mutation site. In
one such
embodiment of the present invention a 2'-0-methyl ribonucleoside is placed
immediately 5' to the RNA base within the primer (see Example 22).
[0167] The sensitivity of the assay can also be increased through
incorporation of
nuclease resistant analogs into the primer on the 3'-side of the base over the
mutation
site. Such nuclease resistant analogs include, but are not limited to,
phosphorothioates,
phosphorodithioates, methylphosphonates and abasic residues such as a C3
spacer. In
one such embodiment of the present invention, phosphorothioate intemucleotide
linkages are incorporated at each position from the RNA base over the mutation
site to
the 3 '-end of the primer. In yet another such embodiment phosphorothioatc
linkages or
phosphoroditioate are incorporated at all positions from the base on the 3'-
side of the
RNA residue to the 3'-end of the primer. In yet another such embodiment a
single
phosphorothioate or phosphorodithioates is introduced on the 3'-side of the
residue
immediately downstream from the RNA base within the primer. In one embodiment,
the phosphorothioate bonds are placed between each monomer 3' to the RNA
monomer
directly over the SNP site, as well as between the RNA monomer and the base 3'
to the
RNA base (see Example 25).
[0168] The assay sensitivity can also be improved by optimizing the
placement of
the 3' blocking group or groups. In one embodiment, a blocking group is placed
internal to the 3' end of the oligonucleotide. In a further embodiment, more
than on
blocking group is placed internal to the 3' terminus. In yet a further
embodiment, an
43
CA 02949315 2016-11-23
RNA monomer sits directly over the SNP site, with a DNA monomer 3' to the RNA
monomer, followed by two C3 spacers, and finally followed by a 3' terminal
base (see
Example 28).
[0169] In one embodiment of the allele-specific PCR, the primers can be
designed
to detect more than one mismatch. For example, the forward primer can detect a
first
mismatch, and the reverse primer could detect a second mismatch. In this
embodiment,
the assay can be used to indicate whether two mismatches occur on the same
gene or
chromosome being analyzed. This assay would be useful in applications such as
determining whether a bacterium of interest is both pathogenic and antibiotic
resistant.
Reverse transcriptase PCR (RT-PCR)
[0170] In yet another embodiment the methods of the present invention can
be used
in coupled reverse transcription-PCR (RT-PCR). In one such embodiment reverse
transcription and PCR are carried out in two disctinct steps. First a cDNA
copy of the
sample mRNA is synthesized using either an oligo dT primer or a sequence
specific
primer. Random hexamers and the like can also be used to prime cDNA synthesis.
The
resulting cDNA is then used as the substrate for PCR employing the blocked
primers
and methods of the present invention.
[0171] Alternatively reverse transcription and PCR can be carried out in a
single
closed tube reaction. In one such embodiment three primers are employed, one
for
reverse transcription and two for PCR. The primer for reverse transcription
binds to the
mRNA 3' to the position of the PCR amplicon. Although not essential, the
reverse
transcription primer can include RNA residues or modified analogs such as
2'-0-methyl RNA bases which will not form a substrate for RNase H when
hybridized
to the mRNA. Preferably an RNase H2 enzyme which has decreased activity at
lower
temperatures is used as the cleaving agent.
[0172] In the three primer RT-PCR assay it is desirable to inhibit the RT-
primer
from participating in the PCR reaction. This can be accomplished by utilizing
an
RT-primer having a lower Tm than the PCR primers so it will not hybridize
under the
PCR conditions. Alternatively, a non-replicable primer incorporating, for
example,
two adjacent C3 spacers can be used as the RT-primer (as in polynomial
amplification,
44
CA 02949315 2016-11-23
sec U.S. Pat. No. 7,112,406). In this case when the cDNA is copied by
extension of the
forward PCR primer it will not include the binding site for the RT-primer.
[0173] In one embodiment, only the reverse PCR primer is blocked utilizing
the
compositions and methods of the present invention. In yet another embodiment
both
the forward and reverse PCR primers are blocked. The reverse PCR primer is
blocked
in the 3 primer RT-PCR assay to prevent it from being utilized for reverse
transcription.
If desired, modified bases such as 2'-0-methyl RNA residues can be
incorporated in
the reverse PCR primer although any such modification must allow the primer
sequence to serve as a template for DNA synthesis and be copied.
[0174] In the two primer RT-PCR assays of the present invention, only the
forward
PCR is blocked. The reverse PCR primer also serves as the RT-primer and
therefore
can not be blocked.
[0175] While not comprehensive, Table 1 illustrates how variations in the
blocking
groups, labeling groups, cleavage site embodiments, modifications to the
cleavage site
or other regions of the oligonucleotide, buffer conditions and enzyme can
further
optimize assay formats depending on their particular application. Examples of
assay
formats and applications include PCR; real-time PCR utilizing double-stranded
DNA-binding dyes such as SYBR Green, 5' nuclease assays (TaqmanTm assays) or
molecular beacons; primer-probe and template-probe assays (see U.S. Patent
Application 2009/0068643); polynomial or linked linear amplification assays;
gene
construction or fragment assembly via PCR; allele-specific PCR and other
methods
used to detect single nucleotide polymorphisms and other variant alleles;
nucleic acid
sequencing assays; and strand displacement amplification. In these various
assays,
cleavage of the primers of the present invention can be used to enhance the
specificity
of the particular reaction.
Table 1: PCR/Primer Extension/Polyamp
'
Primer Blocking Labeling RNase H Cleavage Flanking
Divalent DNA RNase II Sample Use Assay Format
Group Group Site sequence cation Polymerase
modifications
None None RNA None mg2,- Hot Start
RNase HI Genomic Sample Prep No additional probe
1. Single RNA 1. Ab DNA
1. Detection of primer
_____________________ residue _________ ¨ 2.Chemi-
cleavage I
Fluorophore -,. Multiple RNA Nuclease- cally
RNase H2 Coupled A. Fluorescence
resistant linkages I. Non-
amplification
residues modifiedB. Mass Spec
1. thermostable to reverse
C. Electrophoresis
Phosphorothioate 2. Thennostable
transcription 2. Dye-binding assay
2. Dithioate A. Hot Start
A. Sybr Green
Fluorophore/ 3. Methyl- i. Intrinsic
Quencher phosphonate ii. Ab
0
4. Non-nucleotide iii.
Chemically
spacers modified
o
to
B. Non-Hot Start
_______________________________________________________________________________
___________________ l0
Enzyme
Mitochon- Quantification oo.
ko
_______________________________________________________________________________
__ 1. Ilorseradish drial/ ______ of target w
Modification of 3' - peroxidase Modified residues:
Alter- Non-llot RNase 113 and chloroplast nucleic acid With
an internal probe i-,
ix
terminal residue 2. Alkaline 1. 2 adjacent 2' F
native Start other catalysts that DNA sequence 1. Taqmafl
Ni
1. C3 spacer phosphatase residues divalent
cleave RNA/DNA 1. Chromoso- 2. Fluorescence- 0
cation heteroduplexes
mal copy quenched i-,
on)
__/_ mg2,
number
linear probe I
I-
2. mRNA
3. Molecular beacon
1
4. RNase H-cleavable
Ni
_________________________________________ _
Biotin 2'0Me
cDNA Detection of probe w
variant allele
Hapten
1. Deoxigenin
Upstream Antibody Secondary RNase H mutants
RNA Gene/Fragment Tempro Assay
modification mismatches having altered
1. mRNA construction 1. RNase 112-
1. Adjacent to the Mass Tag
cleavage cleavable probe
3'-terminal specificity
0
residue Radiolabel I. Enhanced
i
2. Further upstream 32P, 34C, 3H,
cleavage of 2'-F 1
,
35S, etc. substrates
o
o
4
i
;
46
CA 02949315 2016-11-23
Cycling Probe Reactions
[0176] Cycling probe reactions are another technique for detecting specific
nucleic
acid sequences (see U.S. Pat. No. 5,403,711). The reaction operates under
isothermal
conditions or with temperature cycling. Unlike PCR products accumulate in a
linear
fashion.
[0177] Table 2 illustrates a non-comprehensive set of possible elements of
the
current invention to improve assays based on the cycling probe reaction. New
features
of the invention include 1) use of a hot start RNase H enzyme; 2) cleavage of
novel
sequences by RNase H enzymes (e.g., cleavage of substrates containing
2'-fluoronucleosides by Type II RNascs H); and 3) introduction of
modifications and
secondary mismatches flanking an RNase H cleavage domain to enhance
specificity
and/or suppress nonspecific cleavage reactions. Such modifications and
secondary
mismatches are particularly useful when cleavage is mediated by a Type II
RNase H
and the cleavage domain is a single RNA residue or two adjacent 2'-fluoro
residues.
47
Table 2: Cycling Probe Reaction
c
Primer Labeling RNase. H Flanking Divalent
RNase H Sample Use Assay Format 1
<
,
Extension Group Cleavage Site
sequence mods cation ,
,
. Blocking Group
,
i
None None RNA None Mg2+ RNase H1
Genomic Quantification of Stand-alone i
1. Single RNA DNA
____________________________________________________________________ target
nucleic acid I. Isothermal
Modification of Fluorophore residue Nuclease-resist RNase H2
sequence 2. Temperature cycling
3'- terminal 2. Multiple ant linkages 1. Non-
thermostablc 1. Chromosomal
residue RNA residues 1.
2. Thermostable copy number
1. C3 spacer Phos-phorothi A. Hot Start
2. mRNA
Fluorophore/ oate i.
Intrinsic 0
Quencher 2. Dithioate ii. Ab
Mito-chondr
3.
iii. Chemically tali o
N.)
Methyl-phosph modified
chloroplast l0
tP=
onate B. Non-Hot
Start DNA l0
W
Upstream Enzyme Modified 4. Altemativ RNase H3 and
other Detection of variant Coupled to
ix
modification 1. Horseradish residues: Non-nucleotid
e divalent catalysts that cleave allele Amplification
N.)
1. Adjacent to peroxidase 1. 2 adjacent 2' e
spacers cation +/- RNA/DNA 1. PCR 0
the 2. Alkaline F residues m22
heteroduplexes
2. LCR
cn
3' -terminal phosphatase
3. Polyamp 1
1-,
,
residue
'7
2. Further Biotin 2'0Me
RNase H mutants having cDNA N.)
w
upstream altered
cleavage
Hapten specificity
I. Deoxigenin 1. Enhanced
cleavage of
Antibody Secondary 2'-F
substrates
mismatches
Mass Tag
=
!
Radiolabel
c
32p, 14C, 3H,
I
35S, etc.
<
,
<
i
i
48
CA 02949315 2016-11-23
DNA Ligation Assays
101781 The present invention can also serve to increase the specificity of
DNA
ligation assays. Donor and/or acceptor oligonucleotides of the present
invention can be
designed which bind adjacent to one another on a target DNA sequence and are
modified to prevent ligation. Blocking groups on the acceptor oligonucleotide
useful to
inhibit ligation are the same as those used to prevent primer extension.
Blocking the
donor oligonucleotide can be readily accomplished by capping the 5'-OH group,
for
example as a phosphodiester, e.g..:
0
Other 5' blocking groups include 5'-0-alkyl substituents such as 5'-0-methyl
or
5'-0-trityl groups, 5'-0-heteroalkyl groups such as 5'-OCH2CH2OCH3, 5'-0-aryl
groups, and 5'-0-sily1 groups such as TIPS or TBDMS. A 5' deoxy residue can
also be
used to block ligation.
101791 Sterically bulky groups can also be placed at or near the 5'-end of
the
oligonucleotide to block the ligation reaction. A 5'-phosphate group cannot be
used to
block the 5'-OH as this is the natural substrate for DNA ligase. Only after
hybridization to the target DNA sequence are the blocking groups removed by,
for
example cleavage at an RNase H cleavable domain, to allow ligation to occur.
Preferably cleavage is mediated by an RNase H Type II enzyme, and even more
preferably a thermophilic Type IT RNase H enzyme. More preferably, a
thermophilic
Type II RNase H enzyme which is less active at room temperature than at
elevated
temperature is utilized to mediate cleavage and thereby activation of the
acceptor
and/or donor oligonucleotide. Alternatively, a sequence specific nicking
enzyme, such
as a restriction enzyme, may be utilized to mediate cleavage of the donor
and/or
acceptor oligonucleotide.
49
CA 02949315 2016-11-23
[0180] In a further embodiment, the cleaving reaction is first carried out
at a higher
temperature at which only one of the two oligonucleotides hybridizes to the
target
sequence. The temperature is then lowered, and the second oligonucleotide
hybridizes
to the target, and the ligation reaction then takes place.
[0181] In yet a further embodiment in which there is a cleavage domain
located in
the donor oligonucleotide, this oligonucleotide is not blocked at or near the
5'-end, but
simply has a free 5'-OH. This oligonucleotide cannot serve as a donor in the
ligation
reaction; to do so requires a 5'-phosphate group. Thus, the 5'-end is
functionally
blocked. Cleavage by RNase H generates a 5'-phosphate group allowing the donor
oligonucleotide to participate in the ligation reaction.
[0182] An important advantage of the present invention is that it allows
double
interrogation of the mutation site, and hence greater specificity, than
standard ligation
assays. There is an opportunity for discrimination of a variant allele both at
the
cleavage step and the ligation step.
[0183] Table 3 illustrates a non-comprehensive set of possible elements of
the
current invention to improve oligonucleotide ligation assays.
0
o
m Table 3: Oligonucleotide Ligation Assay
_
m Donor Acceptor Labeling RNase H Flanking sequence
Divalent DNA Ligase RNase H Sample Use Reaction Assay
Fe
Is)
Oligonucleotide Oligonucleotide Group Cleavage Site
mods cation Conditions o
o
Blocking Group Blocking Group
o
H
..._,
_______________________________________________________________________________
__________________________________ cz -
H
_______________________________________________________________________________
__________________________
0 None None None RNA None Mg 2' Hot Start
RNase HI Genomic Quantification RNase H cleavage Stand-alc`z
i
1--, (5'-phosphate) 1. Single
1. Ab DNA of target and DNA ligation 1. Single cycle
o ________________________________________________________ RNA residue
2. Chem-ically nucleic acid at single 2. Linear
tio 5'-OH Modification of Fluorophore 2. Multiple
Nuclease-resistant modified RNase H2 sequence
temperature Amplification
ul (Functional block) 3.-terminal residue RNA
residues linkages I. Non-thermostable I .Chromosomal
3. LCR
1. C3 spacer 1. 2.
Thermostable copy number
Phos-phorothioate A. Hot
Start 2. mRNA
2. Dithioate i.
Intrinsic
Modification of 5'- Fluorophore/ 3. ii. Ab
residue Quencher Methyl-phosphona
iii. Chemically Mito-chondri 0
1. C3 spacer te
modified at.'
0
4. Non-nucleotide B. Non-
Hot Start chloroplast tv
Downstream Upstream Enzyme Modified spacers
4thernativ Non-Hot Start RNase H3 and other DNA Detection of RNase
H cleavage Coupled to to
modification modification 1. residues: e
divalent catalysts that cleave variant at elevated
primer extension itl.
to
1. Adjacent to the 1. Adjacent to the Horse-radish I. 2
adjacent 2' cation +/- RNA/DNA allele temperature 1. PCR U.)
S.-terminal 3'-terminal peroxidase F residues mg2-i
heteroduplexes
(reduced 2. Reverse
Ln
residue residue 2. Alkaline
temperature for transcription
tv
2. Further 2. Further
phosphatase DNA ligation) 3. Polyamp 0
downstream upstream
cs)
i
Biotin 2'0Me RNase H
mutants cDNA ¨,
1¨,
having altered
1 i
Hapten cleavage
specificity tv
LA.)
1. Deoxi- I.
Enhanced cleavage
genin of 2'-F
substrates
Antibody Secondary
mismatches
Mass Tag
Radiolabel
nO
32p, 14c, 3u,
(...)
35, etc.
CA
1,)
0
0
to
7: Ei
4=,
=F
CA
4.-
51
CA 02949315 2016-11-23
Sequencing Reactions
101841 In one embodiment, a method of sequencing a target DNA of interest
is
provided. The method entails
(a) providing a reaction mixture comprising a primer having a cleavage
domain and
a blocking group linked at or near to the 3' end of the primer which prevents
primer
extension, a sample nucleic acid comprising the target DNA sequence of
interest, a
cleaving enzyme, nucleotide triphosphate chain terminators (e.g., 3'
dideoxynucleotide
triphosphates) and a polymerase,
(b) hybridizing the primer to the target nucleic acid to form a double-
stranded
substrate;
(c) cleaving the hybridized primer with the cleaving enzyme at a point
within or
adjacent to the cleavage domain to remove the blocking group from the primer;
and
(d) extending the primer with the polymerase.
[0185] In one embodiment, the invention is used in a "next generation"
sequencing
platform. One type of next generation sequencing is "sequencing by synthesis",
wherein genomic DNA is sheared and ligated with adapter oligonucleotides or
amplified by gene-specific primers, which then are hybridized to complementary
oligonucleotides that are either coated onto a glass slide or are placed in
emulsion for
PCR. The subsequent sequencing reaction either incorporates dye-labeled
nucleotide
triphosphates or is detected by chemiluminescence resulting from the reaction
of
pyrophosphate released in the extension reaction with ATP sulfurylase to
generate ATP
and then the ATP-catalyzed reaction of luciferase and its substrate luciferin
to generate
oxyluciferin and light.
[01861 A second type of next generation sequencing is "sequencing by
ligation",
wherein four sets of oligonucleotides are used, representing each of the four
bases. In
each set, a fluorophore-labeled oligonucleotide of around 7 to 11 bases is
employed in
which one base is specified and the remaining are either universal or
degenerate bases.
If, for example, an 8-base oligonucleotide is used containing 3 universal
bases such as
inosine and 4 degenerate positions, there would be 44 or 256 different
oligonucleotides
in each set each with a specified base (A, T, C or G) at one position and a
fluorescent
52
CA 02949315 2016-11-23
label attached to either the 5'- or 3'-end of the molecule or at an internal
position that
does not interfere with ligation. Four different labels are employed, each
specific to
one of the four bases. A mixture of these four sets of oligonucleotides is
allowed to
hybridize to the amplified sample DNA. In the presence of DNA ligase the
oligonucleotide hybridized to the target becomes ligated to an acceptor DNA
molecule.
Detection of the attached label allows the determination of the corresponding
base in
the sample DNA at the position complementary to the base specified within the
oligonucleotide.
[0187] In one
embodiment of the present invention, a donor oligonucleotide of
about 7-11 bases contains a specified base at the 5' end of the
oligonucleotide. The
remaining bases are degenerate or universal bases, and a label specific to the
specified
base is incorporated on the 3' side of the specified base. The 3' end of the
probe is
irreversibly blocked to prevent the donor oligonucleotide from also acting as
an
acceptor. In some cases this may be accomplished by the labeling group. The
second
base from the 5' end of the oligonucleotide, i.e., the residue next to the
specified base is
a degenerate mixture of the 4 RNA bases. Alternatively, any anaolog recognized
by
RNase 1-12, such as a 2'-fluoronucleoside may be substituted at this position.
A
universal base such as riboinosine or ribo-5-nitroindole, may also be
incorporated at
this location. The probe first hybridizes to the target sequence and becomes
ligated to
the acceptor DNA fragment as in the standard sequencing by ligation reaction.
After
detection of the specified base, RNase H2 is added which cleaves the probe on
the
5'-side of the RNA residue leaving the specified base attached to the 3' end
of the
acceptor fragment. The end result is that the acceptor fragment is elongated
by one
base and now is in position to permit the determination of the next base
within the
sequence. The cycle is repeated over and over, in each case moving the
position of
hybridization of the donor oligonucleotide one base 3' down the target
sequence. The
specificity is increased compared to traditional sequencing by ligation
because the
specified base is always positioned at the junction of the ligation reaction.
101881 The donor
oligonucleotide probe can optionally contain universal bases
including, but not limited to, 5-nitroindole, ribo-5'-nitroindole, 2'-0-
methyl-
5-nitroindole, inosine, riboinosine, 2'-0-methylriboinosine and 3-
nitropyrrole. This
reduces the number of different oligonucleotides in each set required for the
assay by a
53
CA 02949315 2016-11-23
factor of four for every degenerate position on the probe substituted with a
universal
base. The method can also include a capping step between the ligation reaction
and the
RNase H2 cleaving step. The capping reaction can be performed by introducing a
DNA
polymerase and a chain terminator, thereby capping any of the acceptor
fragment
molecules that did not ligate with a donor oligonucleotide probe in the
previous step.
101891 In the above example the ligation reactions and hence the sequencing
readout proceeds in the 5'- to 3'-direction one base at a time. Alternatively
the donor
oligonucleotide can be designed so that two bases are determined in each
cycle. In this
case the first two bases on the 5'-end of the donor oligonucleotide are
specified (for
example, pA-C-R-N-N-N-I-I-X, where R = a degenerate mixture of all 4 RNA
bases, N
= a degenerate DNA base, I = inosine, and X is a fluorophore). As in all cases
there is a
5'-phosphate (p) to permit ligation of the donor oligonucleotide to the
acceptor.
Sixteen such oligonucleotide sets are required, one for each of the sixteen
possible
dinucleotides. Each of the sixteen can be labeled with a different
fluorophore.
Alternatively ligation reactions can be carried out with 4 separate pools each
having
four such sets of oligonucleotides. In that case, only four different
fluorophores are
required.
101901 In another embodiment for sequencing in the 5'- to 3'-direction a
donor
oligonucleotide of the following type can be used: pA-N-R-N-N-N-1-1-X wherein
p, N,
R, I and X are as defined in the previous example. One base is determined at
each cycle
but at alternate positions: 1, 3, 5, etc. This may be adequate for
identification of the
sequence if compared to a reference database. If desired, the remaining bases
(positions 2, 4, 6, etc.) can be determined by repeating the sequencing
reaction on the
same template with the original acceptor oligonucleotide shifted one base
upstream or
downstream. In a related example a donor oligonucleotide of the following type
can be
used: p-A-F-FN-N-N-I-I-X wherein p, N, I and X are as defined above and F is a
degenerate mixture of all four 2'-fluoronucleosides. Following ligation,
cleavage by
RNase H2 results in the addition of two bases to the 3 '-end of the acceptor
(i.e., AF).
After the next ligation reaction, the sequence at the 3'-end of the acceptor
would
be ...A-F-S-F-F-N-N-N-I-I-X where S is the specified base at position 3, and X
would
be a different fluorophore from the previous cycle if the specified base were
not A.
Cleavage with RNase H2 next occurs between the two 2'-fluororesidues. Cleavage
by
54
CA 02949315 2016-11-23
RNase H2 at the isolated 2'-fluororesiduc occurs much more slowly and can be
avoided
by adjusting the RNase H2 concentration and reaction time.
[0191] A variant of the above method can be performed in which sequencing
proceeds in the 3'- to 5'-direction. In this case an acceptor oligonucleotide
is added at
each cycle as in the following structure: X-I-I-N-N-N-F-F-S-OH wherein the
specified
base (S) is at the 3'-end of the oligonucleotide. The 5'-end is blocked to
prevent the
oligonucleotide from acting as a donor. Cleavage by RNase H2 leaves the
sequence
pF-S at the 5'-end of the donor fragment which is prepared for the next
sequencing
cycle. A capping step can be included in the cycle before the cleavage
reaction using a
phosphatase to remove the 5'-phosphate of the donor oligonucleotide if
ligation to the
acceptor failed to occur.
101921 In a further embodiment, the invention provides an improvement for
DNA
sequencing using ribotriphosphates (or alternative analogs which provide a
substrate
for RNase H2, such as 2'-fluoronucleoside triphosphates) in conjunction with a
fluorescently labeled primer. Similar to traditional sequencing methods known
in the
art, the triphosphate residue would be incorporated by a DNA polymerase. The
concentration of the ribo triphosphatc, or the alternative analog providing a
substrate
for RNase H2, is adjusted to a concentration such that on average one such
base is
incorporated randomly within each extension product produced by the
polymerase.
The nested family of fragments originating from the primer is generated by
cleavage
with RNase H2 and then separated by electrophoresis as in standard DNA
sequencing
methods. Alternatively, multiple RNA residues or modified nucleosides such as
2'-fluoronucleosides may be incorporated into the extension product and the
subsequent digestion with RNase H2 is limited so that on average each strand
is cut
only once. Four separate reactions are run, each substituting one of the bases
with a
different ribotriphosphate (A, C, T or G) or other RNase H2 cleavable analog.
In this
assay, use of expensive fluorcscently labeled dideoxy triphosphate chain
terminators is
obviated.
[0193] In another embodiment of the present invention, an improved method
for
oligonucleotide synthesis is provided. Using similar techniques as described
above, a
composition acting as a donor oligonucleotide can be ligated to an acceptor
fragment in
order to add additional bases to the 3'-end of the acceptor fragment. It is
the acceptor
CA 02949315 2016-11-23
fragment that is the growing polynucleotide undergoing synthesis. In this
case, the
composition of the donor fragment is preferably a single-stranded
oligonucleotide that
forms a hairpin to provide a double-stranded region with an overhang of about
1-8
bases on the 3'-end. The base at the 5' end would be the desired base to add
to the
growing acceptor fragment. For synthesis of a polynucleotide containing all
four bases
(A, C, T and G), four different donor fragments are employed which can have
the
identical sequence except varying in the 5' base. Preferably the donor is
blocked at the
3'-end so it cannot react as an acceptor. The blocking group placed at or near
the 3'-end
of the donor can be a label to allow monitoring of the reaction. Four
different labels can
be used corresponding to the four different bases at the 5'-end of the donor.
The base
adjacent to the desired base at the 5'-end is a RNA base or an alternative
analog such as
a 2'-fluoronucleoside which provides a substrate for RNase H2. The overhang at
the
3'-end can be random (degenerate) bases or universal bases or a combination of
both.
The donor fragment binds to the acceptor fragment, through hybridization of
the 3'-end
of the acceptor to the 3'-overhang of the donor oligonucleotide. A DNA ligase
enzyme
is then used to join the two fragments. Next a Type II RNase H is used to
cleave the
product on the 5'-side of the RNase H2 cleavage site, transferring the 5' base
of the
donor to the 3'-end of the acceptor. Optionally, a third step can be included
in the cycle
between the ligase and RNase H2 cleavage reactions in which molecules of the
growing
polynucleotide chain which may have failed to ligate are capped by reaction
with a
dideoxynucleotide triphosphate (or other chain terminator) catalyzed by a DNA
polymerasc. In one embodiment the DNA polymerase is a dcoxynucleotide terminal
transferasc. The cycle is repeated, and the acceptor fragment can continue to
be
extended in a 5' to 3' direction. To facilitate isolation of the growing
polynucleotide at
each step the acceptor can be attached to a solid support such as controlled
pore glass or
polystyrene
[0194] Similar to the
sequence method described above, a donor oligonucleotide
can be used to add two bases to the 3'-end of the acceptor oligonucleotide at
each cycle.
In this case the RNase H2 cleavable residue would be positioned 3' from the 5'
end of
the donor. This enzymatic synthesis method is particularly advantageous for
synthesis
of longer DNA molecules. The hairpin reagents corresponding to each base can
be
collected for reuse in further cycles or additional syntheses. Because the
system does
not use organic solvents, waste disposal is simplified.
56
CA 02949315 2016-11-23
Kits of the Present Invention
[0195] The present
invention also provides kits for nucleic acid amplification,
detection, sequencing, ligation or synthesis that allow for use of the primers
and other
novel oligonucleotides of the present invention in the aforementioned methods.
In
some embodiments, the kits include a container containing a cleavage compound,
for
example a nicking enzyme or an RNase H enzyme; another container containing a
DNA polymerase and/or a DNA ligase and preferably there is an instruction
booklet for
using the kits. In certain embodiments, the kits include a container
containing both a
nicking enzyme or an RNase H enzyme combined with a DNA polymerase or DNA
ligase. Optionally, the modified oligonucleotides used in the assay can be
included
with the enzymes. The cleavage enzyme agent, DNA polymerase and/or DNA ligase
and oligonucleotides used in the assay are preferably stored in a state where
they
exhibit long-term stability, e.g., in suitable storage buffers or in a
lyophilized or freeze
dried state. In addition, the kits may further comprise a buffer for the
nicking agent or
RNase H, a buffer for the DNA polymerase or DNA ligase, or both buffers.
Alternatively, the kits may further comprise a buffer suitable for both the
nicking agent
or RNase H, and the DNA polymerase or DNA ligase. Buffers may include RNasin
and
other inhibitors of single stranded ribonucleases. Descriptions of various
components
of the present kits may be found in preceding sections related to various
methods of the
present invention.
[0196] Optionally, the
kit may contain an instruction booklet providing information
on how to use the kit of the present invention for amplifying or ligating
nucleic acids in
the presence of the novel primers and/or other novel oligonucleotides of the
invention.
In certain embodiments, the information includes one or more descriptions on
how to
use and/or store the RNase H, nicking agent, DNA polymerase, DNA ligase and
oligonucleotides used in the assay as well as descriptions of buffer(s) for
the nicking
agent or RNase H and the DNA polymerase or DNA ligasc, appropriate reaction
temperature(s) and reaction time period(s), etc.
[0197] Accordingly, in
one embodiment, a kit for the selective amplification of a
nucleic acid from a sample is provided. The kit comprises
(a) a first and a
second oligonucleotide primer, each having a 3' end and 5' end,
wherein each oligonucleotide is complementary to a portion of a nucleic acid
to be
57
CA 02949315 2016-11-23
amplified or its complement, and wherein at least one oligonucleotide
comprises a
RNase H cleavable domain, and a blocking group linked at or near to the 3' end
of the
oligonucleotide to prevent primer extension and/or to prevent the primer from
being
copied by DNA synthesis directed from the opposite primer;
(b) an RNase H enzyme; and
(c) an instruction manual for amplifying the nucleic acid.
The kit may optionally include a DNA polymerase.
[0198] In a further embodiment, the kit for selective amplification of a
nucleic acid
includes an oligonucleotide probe having a 3' end and a 5' end comprising an
RNase H
cleavable domain, a fluorophore and a quencher, wherein the cleavable domain
is
positioned between the fluorophore and the quencher, and wherein the probe is
complementary to a portion of the nucleic acid to be amplified or its
complement.
[0199] In yet another embodiment, the present invention is directed to a
kit for the
ligation of an acceptor oligonucleotide and a donor oligonucleotide in the
presence of a
target nucleic acid sequence. The kit comprises
(a) a donor oligonucleotide and an acceptor oligonucleotide in which one or
both of
the oligonucleotides comprise an RNase H cleavable domain and a blocking group
preventing ligation;
(b) an RNase H enzyme; and
(c) an instruction manual for ligating the acceptor and donor
oligonucleotides in the
presence of a target nucleic acid sequence.
[0200] In a further embodiment, the kit may optionally include a DNA ligase
enzyme.
[0201] In a further ligation kit embodiment, the donor oligonucleotide
contains an
RNase H cleavage domain, but lacks a blocking group at or near the 5'-end and
instead
has a free 5'-OH.
Examples
[0202] The present invention is further illustrated by reference to the
following
Examples. However, it should be noted that these Examples, like the
embodiments
58
CA 02949315 2016-11-23
described above, are illustrative and are not to be construed as restricting
the enabled
scope of the invention in any way.
EXAMPLE 1 ¨ Cloning of codon optimized RNase 112 enzymes from
thermophilic organisms
[0203] This example describes the cloning of codon optimized RNase H2
enzymes
from thermophilic organisms.
[0204] To search for functional novel RNase H2 enzymes with potentially new
and
useful activities, candidate genes were identified from public nucleotide
sequence
repositories from Archaeal hyperthermophilic organisms whose genome sequences
had
previously been determined. While RNase 112 enzymes do share some amino acid
homology and have several highly conserved residues present, the actual
homology
between the identified candidate genes was low and it was uncertain if these
represented functional RNase H2 enzymes or were genes of unknown function or
were
non-functional RNase H2 genes. As shown in Table 4, five genes were selected
for
study, including two organisms for which the RNase H2 genes have not been
characterized and three organisms to use as positive controls where the RNase
112
genes (rnhb) and functional proteins have been identified and are known to be
functional enzymes. Although two uncharacterized predicted rnhb genes were
selected
for this initial study, many more Archaeal species have had their genome
sequences
determined whose rnhb genes arc uncharacterized which could similarly be
studied.
Table 4. Five candidate RNase H2 (rnhb) genes from thermophilic bacteria
Organism Accession # Length Comments
Pyrococcus 687 bp, 228
AB012613 See References (1-3) below
kodakaraensis AA
Pyrococcus AE010276 675 bp, 224 See Reference (4) below and
,furiosus AA UA20040038366A1
Alethanocaldococcus 693 bp, 230
U67470 See References (5,6) below
jannaschii AA
Pyrococcus 675 bp, 224
AJ248284 uncharacterized
abyssi AA
Sulfo/obus 639 bp, 212
AE006839 uncharacterized
solfataricus AA
Bp = base pairs; AA = amino acids
59
CA 02949315 2016-11-23
References 1-6: 1) Haruki, M., Hayashi, K., Kochi, T., Muroya, A., Koga, Y.,
Morikawa, M., Imanaka, T. and Kanaya, S. (1998) Gene cloning and
characterization
of recombinant RNase HIT from a hyperthermophilic archaeon. .1 Bacteriol, 180,
6207-6214; 2) Haruki, M., Tsunaka, Y., Morikawa, M. and Kanaya, S. (2002)
Cleavage
of a DNA-RNA-DNA/DNA chimeric substrate containing a single ribonucleotide at
the DNA-RNA junction with prokaryotic RNases HIT. FEBS Lett, 531, 204-208; 3)
Mukaiyama, A., Takano, K., Haruki, M., Morikawa, M. and Kanaya, S. (2004)
Kinetically robust monomeric protein from a hyperthermophile. Biochemistry,
43,
13859-13866 4) Sato, A., Kanai, A., Itaya, M. and Tomita, M. (2003)
Cooperative
regulation for Okazaki fragment processing by RNase HIT and FEN-1 purified
from a
hyperthermophilic archaeon, Pyrococcus furiosus. Biochem Biophys Res Commun,
309,
247-252; 5) Lai, B., Li, Y., Cao, A. and Lai, L. (2003) Metal ion binding and
enzymatic
mechanism of Methanococcus jannaschii RNase HII. Biochemistry, 42, 785-791;
and 6)
Lai, L., Yokota, H., Hung, L.W., Kim, R. and Kim, S.H. (2000) Crystal
structure of
archaeal RNase HIT: a homologue of human major RNase H. Structure, 8, 897-904.
102051 The predicted physical properties of the proteins encoded by the
rnhb genes
listed above are shown in Table 5 (Pace, C.N. et al., (1995) Protein Sci., 4,
p.2411).
Table 5. Characteristics of five RNase H2 enzymes
# residues
Molecules/lag e 280 nm
Organism Mol. weightTrp, Tyr, -1
protein Nricm
Cys
Pyrococcus
25800.5 2.3E13 1, 7, 0 15930
kodakaren.sis
Pyrococcus
25315.2 2.4E13 2, 8, 0 22920
fUriosus
Alethanocaldococcus
26505.8 2.3E13 1, 9, 3 19285
jannaschii
Pyrococcus
25394.2 2.4E13 3, 7, 0 26930
abysii
Sullblohus
23924.8 2.5E13 3, 10, 0 31400
solfatarieus
102061 The amino acid similarity between RNasc H2 enzymes (or candidate
enzymes) from different Archaeal species within this set of 5 sequences ranges
from
34% to 65%. An amino-acid identity matrix is shown in Table 6 below.
CA 02949315 2016-11-23
Table 6. Amino acid identity between five Archaeal RNase H2 proteins
P. kod. P. fur. M. jann. P. ab. S. soli
P. kodakarensis 0.570 0.595 0.358 0.333
P. fitriosus 0.570 0.654 0.410 0.362
M. jannaschii 0.595 0.654 0.380 0.363
P. abysii 0.358 0.410 0.380 0.336
S. solfataricus 0.333 0.362 0.363 0.336
[0207] Codons of the native gene sequence were optimized for expression in
E. colt
using standard codon usage tables. The following sequences were assembled and
cloned into plasmids as artificial genes made from synthetic oligonucleotides
using
standard methods. DNA sequence identity was verified on both strands.
Sequences of
the artificial DNA constructs are shown below. Lower case letters represents
linker
sequences, including a Barn HI site on the 5' -end and a Hind III site on the
3'-end.
Upper case letters represents coding sequences and the ATG start codons are
underlined.
[0208] SEQ ID NO: 1 ¨ codon optimized rnhb gene from Pyrocoecus
kodakaraensis
ggatccgATGAAGATTGCTGGCATCGATGAAGCCGGCCGTGGCCCGGTAATTGGTCC
AATGGTTATCGCTGCGGTAGTCGTGGACGAAAACAGCCTGCCAAAACTGGAAGAGCT
GAAAGTGCGTGACTCCAAGAAACTGACCCCGAAGCGCCGTGAAAAGCTGTTTAACGA
AATTCTGGGTGTCCTGGACGATTATGTGATCCTGGAGCTGCCGCCTGATGTTATCGG
CAGCCGCGAAGGTACTCTGAACGAGTTCGAGGTAGAAAACTTCGCTAAAGCGCTGAA
TTCCCTGAAAGTTAAACCGGACGTAATCTATGCTGATGCGGCTGACGTTGACGAGGA
ACGTTTTGCCCGCGAGCTGGGTGAACGTCTGAACTTTGAAGCAGAGGTTGTTGCCAA
ACACAAGGCGGACGATATCTTCCCAGTCGTGTCCGCGGCGAGCATTCTGGCTAAAGT
CACTCGTGACCGTGCGGTTGAAAAACTGAAGGAAGAATACGGTGAAATCGGCAGCGG
TTATCCTAGCGATCCTCGTACCCGTGCGTTTCTGGAGAACTACTACCGTGAACACGG
TGAATTCCCGCCGATCGTACGTAAAGGTTGGAAAACCCTGAAGAAAATCGCGGAAAA
AGTTGAATCTGAAAAAAAAGCTGAAGAACGTCAAGCAACTCTGGACCGTTATTTCCG
TAAAGTGaagctt
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CA 02949315 2016-11-23
[0209] SEQ ID NO: 2 ¨ codon optimized mhb gene from Pyrococcus furiosus
ggat ccgATGAAGATTGGTGGCATCGACGAAGCCGGCCGTGGTCCGGCGATCGGTCC
GCTGGTAGTAGCTACTGTTGTAGTGGATGAAAAAAACATCGAAAAACTGCGTAACAT
CGGCGTAAAAGACTCCAAACAGCTGACGCCGCACGAACGTAAAAACCTGTTTTCCCA
GATCACCTCCATTGCGGATGATTACAAGATCGTAATCGTGTCT CC GGAAGAAAT TGA
CAACCGTAGCGGTACCATGAACGAGCT GGAAGT TGAAAAAT TCGCGCTGGCGCTGAA
CTC TO T GCAGAT CAAGCCGGC TOT GAT CTACGCAGACGCAGCAGATGT T GAT GCAAA
CO GC T T CGCATCCC T GATC GAAC GT CGCC T GAAC TATAAAGC CAAAAT CAT CGCGGA
ACACAAAGCAGAC GCAAAG TAO CC GG TCGT T TOT GC GGCGAGCATTCT GGC GAAGGT
T GT GCGTGACGAAGAAATC GAAAAGC TGAAAAAGCAATATGGCGAC T T TGGCAGCGG
T TACC C GAGC GAC C C GAAAAC GAAGAAAT GGC T GGAGGAG TAT TACAAGAAACATAA
CAGC T T CCCACCGATCGT TCGTCGTACGTGGGAAACTGTCCGCAAAAT TGAAGAGTC
CATCAAAGCCAAAAAGTCCCAGCTGACCCTGGATAAATTCTTCAAGAAACCGaagct
[0210] SEQ ID NO: 3 ¨ codon optimized mhb gene from Methanocaldococcus
jannaschii
qgat ccgATGATTATCATTGGTATCGATGAAGCTGGCCGTGGTCCTGTACTGGGCCC
GAT GGT TG TAT GT GC GT TO GC TAT CGAGAAGGAACGT GAAGAAGAAC TGAAAAAGC T
GGGC G T TAAAGAT TO TAAAGAAC T GAO GAAGAATAAAC GC GC G TAO C T GAAAAAGC T
GC T GGAGAACC TGGGCTACGTGGAAAAGCGCATCC TGGAGGC TGAGGAAAT TAACCA
GC TGATGAACAGCAT TAACCTGAACGACAT T GAAATCAACGCATTCAGCAAGGTAGC
TAAAAACCTGATCGAAAAGCTGAACATTCGCGACGACGAAATCGAAATCTATATCGA
CGCTTGTTCTACTAACACCAAAAAGT TCGAAGACTCT TTCAAAGATAAAATCGAAGA
TAT CAT TAAAGAAC GCAATC T GAATAT CAAAAT CAT T GC CGAACACAAAGCAGAC GC
CAAGTACCCAGTAGTGTCTGCGGCGAGCAT TATCGCGAAAGCAGAACGCGACGAGAT
CATCGAT T AT TACAAGAAAAT C TACGGTGACATCGGC TCTGGCTACCCATC TGACCC
GAAAACCATCAAATTCCTGGAAGAT TACT T TAAAAAGCACAAGAAACTGCCGGATAT
C GC TO GCAC T CAC T GGAAAACCT GCAAACGCATCCT GGACAAATC TAAACAGAC TAA
AC TGAT TAT CGAAa agc t t
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CA 02949315 2016-11-23
[0211] SEQ ID NO: 4 ¨ codon optimized rnhb gene from Pyrococcus abysii
ggatccgATGAAAGTTGCAGGTGCAGATGAAGCTGGTCGTGGTCCAGTTATTGGTCC
GCTGGTTATTGTTGCTGCTGTTGTGGAGGAAGACAAAATCCGCTCTCTGACTAAGCT
GGGTGTTAAAGACTCCAAACAGCTGACCCCGGCGCA]CGTGAAAAACTGTTCGATGA
AATCGTAAAAGTACTGGATGATTACTCTGTGGTCATTGTGTCCCCGCAGGACATTGA
CGGTCGTAAGGGCAGCATGAACGAACTGGAGGTAGAAAACTTCGTTAAAGCCCTGAA
TAGCCTGAAAGTTAAGCCGGAAGTTATTTACATTGATTCCGCTGATGTTAAAGCTGA
ACGTTTCGCTGAAAACATTCGCAGCCGTCTGGCGTACGAAGCGAAAGTTGTAGCCGA
ACATAAAGCGGATGCGAAGTATGAGATCGTATCCGCAGCCTCTATCCTGGCAAAAGT
TATCCGTGACCGCGAGATCGAAAAGCTGAAAGCCGAATACGGTGATTTTGGTTCCGG
TTACCCGTCTGATCCGCGTACTAAGAAATGGCTGGAAGAATGGTATAGCAAACACGG
CAATTTCCCGCCGATCGTGCGTCGTACTTGGGATACTGCAAAGAAAATCGAAGAAAA
ATTCA.AACGTGCGCAGCTGACCCTGGACAACTTCCTGAAGCGTTTTCGCAACaagct
[0212] SEQ ID NO: 5 ¨ codon optimized rnhb gene from Sulfblobus
soljataricus
ggatccgATGCGCGTTGGCATCGATGAAGCGGGTCGCGGTGCCCTGATCGGCCCGAT
GATTGTTGCTGGTGTTGTAATCTCTGACACTAAACTGAAGTTTCTGAAAGGCATCGG
CGTAAAAGACTCTAAACAGCTGACTCGCGAGCGTCGTGAAAAGCTGTTTGATATTGT
TGCTAACACTGTGGAAGCATTCACTGTCGTTAAAGTTTTCCCTTATGAAATCGACAA
CTATAACCTGAATGACCTGACCTACGACGCAGTTTCTAAAATCATCCTGAGCCTGTC
TAGCTTTAACCCAGAAATTGTAACGGTTGATAAAGTGGGCGATGAGAAACCGGTTAT
CGAACTGATTAATAAGCTGGGCTACAAAAGCAACGTCGTACACAAGGCAGATGTACT
GTTTGTAGAAGCCTCCGCTGCTAGCATCATTGCGAAAGTTATTCGTGATAACTACAT
TGACGAACTGAAACAAGTATACGGTGACTTTGGTAGCGGTTACCCAGCTGATCCTCG
CACTATCAAATGGCTGAAATCTTTCTACGAAAAGAATCCGAATCCGCCGCCAATCAT
TCGTCGTTCCTGGAAGATTCTGCGTTCTACCGCCCCGCTGTATTACATTTCCAAAGA
AGGTCGCCGTCTGTGGaagctt
EXAMPLE 2 ¨ Expression of recombinant RNase H2 peptides
[0213] The following example demonstrates the expression of recombinant
RNase
H2 peptides.
[0214] The five synthetic gene sequences from Example 1 were subcloned
using
unique Barn HI and Hind III restriction sites into the bacterial expression
vector
pET-27b(+) (Novagen, EMD Biosciences, La Jolla, CA). This vector places six
histidine residues (which together comprise a "His-tag") at the carboxy
terminus of the
63
CA 02949315 2016-11-23
expressed peptide (followed by a stop codon). A "His-tag" permits use of
rapid, simple
purification of recombinant proteins using Ni affinity chromatography, methods
which
are well known to those with skill in the art. Alternatively, the synthetic
genes could be
expressed in native form without the His-tag and purified using size exclusion
chromatography, anion-exchange chromatography, or other such methods, which
are
also well known to a person of ordinary skill in the art.
102151 BL21(DE3) competent cells (Novagen) were transformed with each
plasmid and induced with 0.5 mM isopropyl-I3-D-thio-galactoside (IPTG) for 4.5
hours
at 25 C. For all clones, 5 mL of IPTG induced culture was treated with
Bugbustee)
Protein Extraction Reagent and Benzonase(R Nuclease (Novagen) to release
soluble
proteins and degrade nucleic acids according to the manufacturer's
instructions. The
recovered protein was passed over a Ni affinity column (Novagen) and eluted
with
buffer containing 1M imidazole according to protocols provided by the
manufacturer.
102161 Both "total" and "soluble" fractions of the bacterial lysate were
examined
using SDS 10% polyacrylamide gel electrophoresis. Proteins were visualized
with
Coomassie Blue staining. Following IPTG induction, large amounts of
recombinant
proteins were produced from all 5 Archaeal RNase H2 synthetic genes. Using
this
method of purification, protein was recovered in the soluble fraction for 4
enzymes,
Pyrococcus kotiakaraensis, Pyrococcus .furiosus, Methanocaldococcus
jannaschii, and
Pyrococcus abyssi. No soluble protein was recovered for SulfiVobus
solfataricus
RNase H2 using this lysis procedure. Examples of induced RNase H2 proteins are
shown in Figs. 4A and 4B.
102171 Improved methods to produce and purify the recombinant proteins were
developed to produce small scale amounts of the proteins for characterization
as
follows. To maximize the amount of soluble protein obtained for each clone, an
induction temperature of 37 C is used for 6 hours. For Pyrococcus
kodakaraensis,
Methanocaldococcus jannaschii, and Sulfolobus solfatarictts, CelLyticTM B 10x
lysis
reagent (Sigma-Aldrich, St. Louis, MO) is used for lysis. A 10 fold dilution
in 500 mM
NaCl, 20 mM TrisHCI, 5 mM imidazole, pH 7.9 is made and 10 mL is used per 0.5
g of
pelleted bacterial paste from induced cultures. For Pyrococcus fitriosus and
Pyrococcus abyssi, 5 mL of Bugbuster(R Protein Extraction Reagent (Novagen)
per 100
mL of induced culture is used for cell lysis. In addition, per 100 mL induced
culture for
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CA 02949315 2016-11-23
all clones, 5KU rLysozymeTm (Novagen) and 250U DNase I (Roche Diagnostics,
Indianapolis, IN) is used to enhance bacterial cell lysis and decrease the
viscosity of the
solution. Following centrifugation to remove cell debris, the lysates are
heated for 15
minutes at 75 C to inactive the DNase I and any other cellular nucleases
present. The
lysates are then spun at 16,000 x g for 20 minutes to sediment denatured
protein
following heat treatment. The centrifugation step alone provides a large
degree of
functional purification of the recombinant thermostable enzymes.
102181 The resulting soluble supernatant is passed over a Ni affinity
column
containing His Bind Resin (Novagen) and eluted with an elution buffer
containing 200
rnM imidazole. The purified protein is then precipitated in the presence of
70%
ammonium sulfate and resuspended in storage buffer (10mM Tris pH 8.0, ImM
EDTA,
100mM NaC1, 0.1% Triton X-100, 50% Glycerol) to concentrate and stabilize the
protein for long term storage. The concentrated protein is dialyzed 2 x 2
hours (x250
volumes each) against the same storage buffer to remove residual salts. The
final
purified protein is stored at -20 C. Using these protocols, for Pyrococcus
abysii, 200
mL of IPTG induced culture yields ¨2 mg of soluble protein. After passing over
a Ni
column, ¨0.7mg of pure protein is recovered. For functional use, the
concentrated
enzyme stocks were diluted in storage buffer and added 1:10 in all enzymatic
reactions
studied. Therefore all reaction buffers contain 0.01 % Triton X-100 and 5%
Glycerol.
[0219] Recombinant protein was made and purified for each of the cloned
RNase
H2 enzymes as outlined above. Samples from Pyrococcus kodakaraensis,
Pyrococcus
fitriosus, Pyrococcus abyssi, and S4folobus solfataricus were examined using
SDS
10% polyacrylamide gel electrophoresis. Proteins were visualized with
Coomassie
Blue staining. Results are shown in Figure 5. If the expression and
purification method
functioned as predicted, these proteins should all contain a 6x Histidine tag,
which can
be detected using an anti-His antibody by Western blot. The gel shown in
Figure 5 was
electroblot transferred to a nylon membrane and a Western blot was performed
using an
anti-His antibody. Results are shown in Figure 6. All of the recombinant
proteins were
recognized by the anti-His antibody, indicating that the desired recombinant
protein
species were produced and purified.
[02201 Large scale preparations of the recombinant proteins can be better
expressed
using bacterial fermentation procedures well known to those with skill in the
art. Heat
CA 02949315 2016-11-23
treatment followed by centrifugation to sediment denatured proteins will
provide
substantial purification and final purification can be accomplished using size
exclusion
or anion exchange chromatography without the need for a His-tag or use of Ni-
affinity
chromatography.
EXAMPLE 3 ¨ RNase 112 activity for the recombinant peptides
[0221] The following
example demonstrates RNase H2 activity for the
recombinant peptides.
[0222] RNase H enzymes
cleave RNA residues in an RNA/DNA heteroduplex. All
RNase H enzymes can cleave substrates of this kind when at least 4 sequential
RNA
residues are present. RNase HI enzymes rapidly lose activity as the RNA
"window" of
a chimeric RNA/DNA species is shortened to less than 4 residues. RNase H2
enzymes,
on the other hand, are capable of cleaving an RNA/DNA heteroduplex containing
only
a single RNA residue. In all cases, the cleavage products contain a 3'-
hydroxyl and a
5'-phosphate (see FIG. 1). When multiple RNA residues are present, cleavage
occurs
between RNA bases, cleaving an RNA-phosphate linkage. When only a single RNA
residue is present, cleavage occurs only with Type II RNase H enzymes. In this
case
cleavage occurs on the 5'-side of the RNA base at a DNA-phosphate linkage (see
FIG.
3). RNase H enzymes
require the presence of a divalent metal ion cofactor. Typically,
RNase HI enzymes require the presence of Mg-' ions while RNase H2 enzymes can
function with any of a number of divalent cations, including but not limited
to Mg,
Mn ,Co'and Ni .
[0223] The recombinant
RNase H2 proteins described in Example 2 were tested for
both types of RNase H activity and were examined for the characteristics
listed above.
102241 Cleavage of a
substrate with multiple RNA bases. The following synthetic
30 bp substrate was used to test the activity of these enzymes for cleavage of
a long
RNA domain. The substrate is an "11-8-11" design, having 11 DNA bases, 8 RNA
bases, and 11 DNA bases on one strand and a perfect match DNA complement as
the
other strand. The oligonucleotides employed are indicated below, where upper
case
letters represent DNA bases and lower case letters represent RNA bases.
66
CA 02949315 2016-11-23
102251 SEQ ID NO: 6
5' -CTCGTGAGGTGaugcaggaGATGGGAGGCG-3'
[02261 SEQ ID NO: 7
5' -CGCCTCCCATCTCCTGCATCACCTCACGAG-3'
[0227] When annealed, these single-stranded (ss) oligonucleotides form the
following "11-8-11" double-stranded (ds) substrate:
102281 SEQ ID NO: 8
5' -CTCGTGAGGTGaugcaggaGATGGGAGGCG-3'
3' -GAGCACTCCACTACGTCCTCTACCCTCCGC-5'
[0229] Aliquots of each of the recombinant protein products were incubated
with
single-stranded or double-stranded oligonucleotide substrates in an 80 iii
reaction
volume in buffer 50 mM NaC1, 10 mM MgCl2, and 10 mM Tris pH 8.0 for 20 minutes
at 45 C or 70 C. Reactions were stopped with the addition of gel loading
buffer
(formamide/EDTA) and separated on a denaturing 7M urea, 15% polyacrylamide
gel.
Gels were stained using GelStarim (Lonza, Rockland, ME) and visualized with UV
excitation. All 5 recombinant peptides showed the ability to cleave an 8 base
RNA
sequence in an RNA/DNA heteroduplex (11-8-11) substrate. Importantly, the
recombinant proteins did not degrade the single stranded RNA-containing
oligonucleotide (SEQ ID No. 6), indicating that a double-stranded substrate
was
required. Further, a dsDNA substrate was not cleaved.
102301 Cleavage was not observed in the absence of a divalent cation (e.g.,
no
activity was observed if Mg- was absent from the reaction buffer). A Mg' +
titration
was performed and high enzyme activity was observed between 2-8 mM MgC12.
Optimal activity was observed between 3-6 mM MgC12. Cleavage activity was also
detected using other divalent cations including Mn' and Co' . In MnC12, good
activity
was seen from 0.3 mM to 10 naM divalent cation concentration. Enzyme activity
was
optimal in the range of 300 nM to 1 mM. For CoC12, activity was seen in the
range of
0.3 mM to 2 mM, with optimal activity in the range of 0.5 - 1 mM. The isolated
enzymes therefore show RNase H activity, and divalent cation requirements that
are
characteristic of the RNase H2 class.
67
CA 02949315 2016-11-23
[0231] Digestion of the 11-8-11 substrate by recombinant RNase H2 enzymes
from
Pyrococcus kodakaraensis, Pyrococcus furiasus, and Pyrococcus abyssi is shown
in
Figure 7.
[0232] Substrate cleavage by RNase H enzymes is expected to result in
products
with a 3'-OH and 5'-phosphate. The identity of the reaction products from the
new
recombinant RNasc H2 proteins was examined by mass spectrometry. Electrospray
ionization mass spectrometry (ESI-MS) has near single Dalton resolution for
nucleic
acid fragments of this size (accuracy of +/- 0.02%). The oligonucleotide 11-8-
11
substrate (SEQ ID No. 8) was examined by ESI-MS both before and after
digestion
with the three Pyrococcus sp. RNase H enzymes. The primary masses observed are
reported in Table 7 along with identification of nucleic acid species
consistent with the
observed masses.
Table 7. Mass of species observed after RNase H2 digestion of SEQ ID No. 8
RNase H2 Predicted Observed
Sequence Treatment Mol Wt Mol Wt
5' CTCGTGAGGTGaugcaggaGATGGGAGGCG 9547 9548
None (control)
3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
5' CTCGTGAGGTGa 3717 3719
us
Pyrococc
5' P-aGATGGGAGGCG 3871 3871
kodakaraensis 3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
5' CTCGTGAGGTGa 3717 3719
Pyrococcus
5' P-aGATGGGAGGCG 3871 3872
furiosus 3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
5' CTCGTGAGGTGa 3717 3719
s
Pyrococcu
5' P-aGATGGGAGGCG 3871 3872
abyssi 3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
[0233] Major species identified are shown. DNA bases are indicated with
upper
case letters, RNA bases are indicated with lower case letters, and phosphate =
Molecular weights arc rounded to the nearest Dalton. In the absence of other
notation,
the nucleic acids strands end in a 5'-hydroxyl or 3'-hydroxyl.
[0234] In all cases, the DNA complement strand was observed intact
(non-degraded). The RNA-containing strands were efficiently cleaved and the
observed masses of the reaction products arc consistent with the following
species
being the primary fragments produced: 1) a species which contained undigested
DNA
residues and a single 3'-RNA residue with a 3'-hydroxyl groups (SEQ ID No. 9),
and 2)
a species with a 5'-phosphate, a single 5'-RNA residue, and undigested DNA
residues
68
CA 02949315 2016-11-23
(SEQ ID No. 10). The observed reaction products are consistent with the known
cleavage properties of both RNase HI and RNase H2 enzymes.
[0235] SEQ ID NO: 9 - 5' CTCGTGAGGTGa 3'
[0236] SEQ ID NO: 10 - 5' P-aGATGGGAGGCG 3'
[0237] Cleavage of a substrate with a single RNA base. RNase H2 enzymes
characteristically cleave a substrate that contains a single RNA residue while
RNasc H1
enzymes cannot. The following synthetic 30 bp substrates were used to test the
activity
of these enzymes for cleavage at a single RNA residue. The substrates are a
"14-1-15"
design, having 14 DNA bases, 1 RNA base, and 15 DNA bases on one strand and a
perfect match DNA complement as the other strand. Four different substrates
were
made from 8 component single-stranded oligonucleotides comprising each of the
4
RNA bases: C, G, A, and U. The oligonucleotides employed are indicated below,
where upper case letters represent DNA bases and lower case letters represent
RNA
bases.
For rC:
[0238] SEQ ID NO: 11
'-CTCGTGAGGTGATGcAGGAGATGGG AGGCG-3'
[0239] SEQ ID NO: 12
5' -CGCCTCCCATCTCCTGCATCACCTCACGAG-3 '
[0240] When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 rC" double-stranded (ds) substrate:
102411 SEQ ID NO: 13
5' -CTCGTGAGGTGATGcAGGAGATGGGAGGCG-3'
3' -GAGCACTCCACTACGTCCTCTACCCTCCGC-5'
For rG:
[0242] SEQ ID NO : 14 -5'-CTCGTGAGGTGATGgAGGAGATGGGAGGCG-3'
69
CA 02949315 2016-11-23
10243] SEQ ID NO: 15 - 5'-CGCCTCCCATCTCCTCCATCACCTCACGAG-3'
102441 When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 rG" double-stranded (ds) substrate:
[0245] SEQ ID NO: 16
5' -CTCGTGAGGTGATGgAGGAGATGGGAGGCG-3'
3' -GAGCACTCCACTACCTCCTCTACCCTCCGC-5'
For rA:
[0246] SEQ ID NO: 17-
'-CTCGTGAGGTGATGaAGGAGATGGGAGGCG-3'
10247] SEQ ID NO: 18 - 5 ' -CGCCTCCCATCTCCTTCATCACCTCACGAG-3'
[0248] When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 TA" double-stranded (ds) substrate:
[0249] SEQ ID NO: 19
5' -CTCGTGAGGTGATGaAGGAGATGGGAGGCG-3'
3' -GAGCACTCCACTACTTCCTCTACCCTCCGC-5'
For rU:
102501 SEQ ID NO : 20
5' -CTCGTGAGGTGATGuAGGAGATGGGAGGCG-3'
102511 SEQ ID NO : 21
5' -CGCCTCCCATCTCCTACATCACCTCACGAG-3'
[0252] When annealed, these single-stranded (ss) oligonucleotides form the
following "14-1-15 rU" double-stranded (ds) substrate:
[0253] SEQ ID NO: 22
5' -CTCGTGAGGTGATGuAGGAGATGGGAGGCG-3'
3' -GAGCACTCCACTACATCCTCTACCCTCCGC-5'
CA 02949315 2016-11-23
[0254] Aliquots of each of the recombinant protein products were incubated
with
the single-stranded and double-stranded oligonucleotide substrates indicated
above in
an 80 pl reaction volume in buffer 50 mM NaC1, 10 mM MgC12, and 10 mM Tris pH
8.0 for 20 minutes at 70 C. Reactions were stopped with the addition of gel
loading
buffer (formamidc/EDTA) and separated on a denaturing 7M urea, 15%
polyacrylamide gel. Gels were stained using GelStarTM (Lonza, Rockland, ME)
and
visualized with UV excitation. All 5 recombinant peptides showed the ability
to cleave
a single RNA base in an RNA/DNA heteroduplex (14-1-15). Each of the 4 RNA
bases
functioned as a cleavable substrate with these enzymes. Importantly, the
recombinant
proteins did not degrade the single stranded RNA-containing oligonucleotides
(SEQ ID
Nos. 11, 14, 17, 20), indicating that a double-stranded substrate was
required. The
isolated enzymes therefore show RNase H2 activity. Titration of divalent
cations was
tested and results were identical to those obtained previously using the 8-11-
8 substrate.
102551 Digestion of the four 14-1-15 substrates (SEQ ID Nos. 13, 16, 19,
22) and
the 11-8-11 substrate (SEQ ID No. 8) by recombinant RNasc H2 enzymes from
Pyrococcus abys,si,Pyrococcus fitriosus, and Alethanocaldococcus jannaschii is
shown
in Figure 8A and from Pyrococcus kodakaraensis in Figure 8B.
102561 Substrate cleavage by RNase H enzymes is expected to result in
products
with a 3'-OH and 5'-phosphate. Further, cleavage of a substrate containing a
single
ribonucleotide by RNase H2 enzymes characteristically occurs at the DNA
linkage
5'-to the RNA residue. The identity of the reaction products using a single
ribonucleotide substrate from the new recombinant RNase H2 proteins was
examined
by mass spectrometry. The oligonucleotide 14-1-15 rC substrate (SEQ ID No. 13)
was
examined by ESI-MS both before and after digestion with the three Pyrococcus
sp.
RNase H2 enzymes and the Methanocaldococcus jannaschii enzyme. The primary
masses observed are reported in Table 8 along with identification of nucleic
acid
species consistent with the observed masses.
71
CA 02949315 2016-11-23
Table 8. Mass of species observed after RNase H2 digestion of SEQ ID No. 13
RNase H2 Predicted
Observed
Treatment Sequence Mol Wt Mol Wt
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 9449 9450
None (control)
3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
P yrococcus 5' CTCGTGAGGTGATG 4334 4335
5' P-cAGGAGATGGGAGGCG 5132 5133
kodakaraensiS 3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
5' CTCGTGAGGTGATG 4334 4335
PyrOCOCCUS IiirlOSUS 5' P-cAGGAGATGGGAGGCG 5132 5132
3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
5' CTCGTGAGGTGATG 4334 4335
Pyrococcus abyssi 5' P-cAGGAGATGGGAGGCG 5132 5133
3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
illethanocaldococcus 5' CTCGTGAGGTGATG 4334 4335
5' P-cAGGAGATGGGAGGCG 5132 5133
jannaschii 3' GAGCACTCCACTACGTCCTCTACCCTCCGC 8984 8984
[0257] Major species identified are shown. DNA bases are indicated with
upper
case letters, RNA bases are indicated with lower case letters, and phosphate =
Molecular weights are rounded to the nearest Dalton. In the absence of other
notation,
the nucleic acids strands end in a 5'-hydroxyl or 3'-hydroxyl.
[0258] In all cases, the DNA complement strand was observed intact
(non-degraded). The RNA-containing strands were efficiently cleaved and the
observed masses of the reaction products are consistent with the following
species
being the primary fragments produced: 1) a species which contained undigested
DNA
residues with a 3'-hydroxyl (SEQ ID No. 23), and 2) a species with a 5'-
phosphate, a
single 5'-RNA residue, and undigested DNA residues (SEQ ID No. 24). The
observed
reaction products are consistent with the known cleavage properties of RNase
H2 class
enzymes.
[0259] SEQ ID NO: 23
5' CTCGTGAGGTGATG 3'
[0260] SEQ ID NO: 24
5' P-cAGGAGATGGGAGGCG 3'
[0261] In summary, the cloned, codon-optimized rnhb genes predicted to
encode
RNase H2 enzymes from 5 Archaeal species all produced recombinant protein
products
which displayed enzyme activities consistent with that expected for members of
the
72
CA 02949315 2016-11-23
RNase H2 family. 1) The enzymes required divalent cation to function (the
experiments presented here were done using Mg' '). Activity is also present
using Mn+4
or Coil ions; 2) Single-stranded nucleic acids are not degraded; 3) Double-
stranded
heteroduplex nucleic acids are substrates where one strand contains one or
more RNA
bases; 4) For substrates containing 2 or more consecutive RNA bases, cleavage
occurs
in a DNA-RNA-DNA chimera between RNA linkages; for substrates containing a
single RNA base, cleavage occurs immediately 5'-to the RNA base in a
DNA-RNA-DNA at a DNA linkage; and 6) Reaction products have a 3-hydroxyl and
'-phosphate.
EXAMPLE 4 ¨ Reaction temperature optimization and thermal stability of
Pyrococcus abyssi RNase H2
[0262] For this example and all subsequent work, the amount of the enzyme
employed was standardized based upon the following unit definition, where:
1 unit is defined as the amount of enzyme that results in the cleavage of 1
nmole of a heteroduplex substrate containing a single rC residue per
minute at 70 C in a buffer containing 4 mM Mg2+ at pH 8Ø
102631 Substrate SEQ ID No. 13 was employed for characterizing RNase H2
enzyme preparation for the purpose of normalizing unit concentration. The
following
standardized buffer was employed unless otherwise noted. "Mg Cleavage Buffer":
4
mM MgC12, 10 mM Tris pH 8.0, 50 mM NaCl, 10 j1g/m1 BSA (bovine serum albumin),
and 300 nM oligo-dT (20mer poly-dT oligonucleotide). The BSA and oligo-dT
serve
to saturate non-specific binding sites on plastic tubes and improve the
quantitative
nature of assays performed.
[0264] Purified recombinant Pyrococcus abys,s1 RNase H2 enzyme was studied
for
thermal stability. Aliquots of enzyme were incubated at 95 'V for various
periods of
time and then used to cleave the single rC containing substrate SEQ ID No. 13.
The
RNA strand of the substrate was radiolabeled with 32P using 6000 Ci/mmol y-32P-
ATP
and the enzyme T4 Polynucleotide Kinase (Optikinase, US Biochemical). Trace
label
was added to reaction mixtures (1:50). Reactions were performed using 100 nM
substrate with 100 microunits (gU) of enzyme in Mg Cleavage Buffer. Reactions
were
73
CA 02949315 2016-11-23
incubated at 70 C for 20 minutes. Reaction products were separated using
denaturing
7M urea, 15% polyacrylamide gel electrophoresis (PAGE) and visualized using a
Packard CycloneTM Storage Phosphor System (phosphorimager). The relative
intensity
of each band was quantified using the manufacturer's image analysis software
and
results plotted as a fraction of total substrate cleaved. Results are shown in
Figure 9.
The enzyme retained full activity for over 30 minutes at 95 C. Activity was
reduced to
50% after 45 minutes incubation and to 10% after 85 minutes incubation.
102651 These results demonstrate that the Pyrococcus abyssi RNase H2 enzyme
is
sufficiently thermostable to survive prolonged incubation at 95 C and would
therefore
survive conditions typically employed in PCR reactions.
102661 The temperature dependence of the activity of the Pyrococcus abyssi
RNase
1-12 enzyme was next characterized. The activity was studied over a 40 C
temperature
range from 30 C to 70 C. The RNA strand of the rC substrate SEQ ID No. 13 was
radiolabeled as described above. Reactions were performed using 100 nM
substrate
with 200 microunits (1.1U) of enzyme in Mg Cleavage Buffer. Reactions were
incubated
at 30 C, 40 C, 50 C, 60 C, or 70 C for 10 minutes. Reactions were stopped with
the
addition of cold EDTA containing formamide gel loading buffer. Reaction
products
were then separated using denaturing 7M urea, 15% polyacrylamidc gel
electrophoresis
(PAGE) and visualized using a Packard CycloneTM Storage Phosphor System
(phosphorimager). The resulting gel image is shown in Figure 10. The relative
intensity of each band was quantified using the manufacturer's image analysis
software
and results plotted as a fraction of total substrate cleaved (see FIG. 11).
The enzyme
shows only ¨0.1% activity at 30 C and does not attain appreciable activity
until about
50 to 60 C.
102671 Therefore, for practical purposes the enzyme is functionally
inactive at
room temperature. Reactions employing this enzyme can therefore be set up on
ice or
even at room temperature and the reactions will not proceed until temperature
is
elevated. If Pyrococcu,s' abyssi RNase H2 cleavage were linked to a PCR
reaction, the
temperature dependent activity demonstrated herein would effectively function
to
provide for a "hot start" reaction format in the absence of a modified DNA
polymerase.
74
CA 02949315 2016-11-23
EXAMPLE 5 ¨ Cleavage at non-standard bases by RNase H2
[0268] The natural biological substrates for RNase HI and RNase H2 are
duplex
DNA sequences containing one or more RNA residues. Modified bases containing
substitutions at the 2'-position other than hydroxyl (RNA) have not been
observed to be
substrates for these enzymes. The following example demonstrates that the
Pyrococcus
abyssi RNase H2 enzyme has activity against modified RNA-containing
substrates.
[0269] The following 14-1-15 substrates containing modified bases were
tested to
determine if RNase H2 could recognize single non-RNA 2'-modified bases as
sites for
cleavage. The modifications are located on the 2' position of the base and
include
locked nucleic acid (LNA), 2'-0-methyl (2'0Me), and 2'-fluoro (2'F); the
single
ribo-C containing substrate was employed as positive control. Hereafter, LNA
bases
will be designated with a "+" prefix (+N), 2'0Me bases will be designated with
a
prefix (mN), 2'F bases will be designated with a "f' prefix (fN), and 2'-amino
bases
with an "a" prefix (aN).
Ribo-C Substrate
[0270] SEQ ID NO: 13
5' -CTCGTGAGGTGATGcAGGAGATGGGAGGCG- 3'
3' -GAGCACTCCACTACGTCCTCTACCCTCCGC- 5 '
LNA-C Substrate
102711 SEQ ID NO: 25
5' -CTCGTGAGGTGATG (+C) AGGAGATGGGAGGCG- 3 '
3' -GAGCACTCCACTAC G TCCTCTACCCTCCGC- 5 '
2'0Me-C Substrate
[0272] SEQ ID NO: 26
5' -CTCGTGAGGTGATG (mC) AGGAGATGGGAGGCG- 3 '
3' -GAGCACTCCACTAC G TCCTCTACCCTCCGC-5 '
2'F-C Substrate
[0273] SEQ ID NO: 27
5' -CTCGTGAGGTGATG ( fC) AGGAGATGGGAGGCG- 3 '
3' -GAGCACTCCACTAC G TCCTCTACCCTCCGC- 5 '
[0274] The above 4 substrates were incubated in an 80 tl reaction volume in
various buffers for 20 minutes at 70 C with the recombinant pyrococcus abyssi
RNase
CA 02949315 2016-11-23
H2 enzyme. Buffers tested included 50 mM NaCl, 10 mM Tris pH 8.0 with either
10
mM MgC12, 10 mM CoC12, or 10 mM MnC12. Reactions were stopped with the
addition of gel loading buffer (formamide/EDTA) and separated on a denaturing
7M
urea, 15% polyacrylamide gel. Gels were stained using GelStarTM (Lonza,
Rockland,
ME) and visualized with LTV excitation. Results arc shown in Figure 12. The
control
substrate with a single ribo-C residue was 100% cleaved. The substrates
containing a
single LNA-C or a single 2'0Me-C residue were not cleaved. However, the
substrate
containing a single 2'-F-C residue was cleaved to a small extent. This
cleavage
occurred only in the manganese containing buffer and was not seen in either
cobalt or
magnesium buffers.
102751 Cleavage at a 2'-F-C base was unexpected. Cleavage of 2'-fluoro
bases was
investigated further using the following substrates.
Ribo-C Substrate
[0276] SEQ ID NO: 13
5' -CTCGTGAGGTGATGcAGGAGATGGGAGGCG- 3 '
3' -GAGCACTCCACTACGTCCTCTACCCTCCGC- 5
2'F-C Substrate
[0277] SEQ ID NO: 27
5' -CTCGTGAGGTGATG ( fC) AGGAGATGGGAGGCG- 3 '
3' -GAGCACTCCACTAC G TCCTCTACCCTCCGC- 5 '
2'F-U Substrate
[0278] SEQ ID NO: 28
5'-CTCGTGAGGTGATG(fU)AGGAGATGGGAGGCG-3'
3'-GAGCACTCCACTAC A TCCTCTACCCTCCGC-5'
2'F-C + 2'FU (fCrU) Substrate
[0279] SEQ ID NO: 29
5'-CTCGTGAGGTGATG(fCfU)GGAGATGGGAGGCG-3'
3'-GAGCACTCCACTAC G A CCTCTACCCTCCGC-5'
[0280] The above 4 substrates were incubated in an 80 ).1.1 reaction volume
in a
buffer containing 50 mM NaCl, 10 mM Tris pH 8.0 and 10 mM MnCl2 for 20 minutes
at 70 C with either the recombinant Pyrococcus abyssi RNase H2 enzyme or the
recombinant Pyrococcus jUriosus RNase H2 enzyme. Reactions were stopped with
the
76
CA 02949315 2016-11-23
addition of gel loading buffer (formamide/EDTA) and separated on a denaturing
7M
urea, 15% polyacrylamide gel. Gels were stained using GelStarTM (Lonza,
Rockland,
ME) and visualized with UV excitation. Results are shown in Figure 13. The
control
substrate with a single ribo-C residue was 100% cleaved. The substrates
containing a
single 2'-F-C or single 2'-F-U residue were cleaved to a small extent. The di-
fluoro
substrate containing adjacent 2'-F-C and 2'-F-U residues (fCfU) was cleaved
nearly
100%. Further, both the Pyrococcus abyssi and Pyrococcus fitriosus RNase H2
enzymes cleaved the modified substrate in an identical fashion. This example
demonstrates that the unexpected cleavage of the IC group was not restricted
to fC but
also occurred with fU. More importantly, a combination of 2 sequential 2'-
fluoro
modified bases was a far better substrate for RNase H2.. This novel cleavage
property
was seen for both the P. abyssi and P. fUriosus enzymes. Cleavage of such
atypical
substrates may be a property common to all Archacal RNasc H2 enzymes.
[0281] The identity of
the cleavage products of the di-fluoro fCfU substrate was
studied using mass spectrometry using the methods described in Example 3.
Using
traditional ribonucicotide substrates, cleavage by RNase H enzymes results in
products
with a 3'-OH and 5 '-phosphate. The fCfU substrate (SEQ ID No. 29) was
examined by
ESI-MS both before and after digestion by the recombinant Pyrococcus abyssi
RNase
H2 enzyme. The primary masses observed are reported in Table 9 along with
identification of nucleic acid species consistent with the observed masses.
Table 9. Mass of species observed after RNase H2 digestion of SEQ ID No. 29
RNase H2 Predicted
Observed
Sequence
Treatment Mol Wt Mol
Wt
5'-CTCGTGAGGTGATG(fCfU)GGAGATGGGAGGCG-3' 9446 9446
None (control) 3'-GAGCACTCCACTAC G A CCTCTACCCTCCGC-5f 8993 8994
5' CTCGTGAGGTGATG(fC) 4642 4643
Pyrococcus 5,
P-(fU)GGAGATGGGAGGCG 4822 4823
abyssi 3' GAGCACTCCACTAC G A CCTCTACCCTCCGC 8993 8994
Major species identified are shown. DNA bases are indicated with upper case
letters,
2'-F bases are indicated as fC or fU, and phosphate = "P". Molecular weights
are
rounded to the nearest Dalton. In the absence of other notation, the nucleic
acids
strands end in a 5'-hydroxyl or 3'-hydroxyl.
[0282] The mass
spectrometry data indicates that digestion of a di-fluoro substrate
such as the fCfU duplex studied above by RNase H2 results in cleavage between
the
two fluoro bases. Further, the
reaction products contain a 3'-hydroxyl and
77
CA 02949315 2016-11-23
5'-phosphate, similar to the products resulting from digestion of RNA
containing
substrates.
[0283] Cleavage of the modified bases was not observed in the absence of a
divalent cation. A titration was performed and enzyme activity was observed
between
0.25-10 mM MnC12 and 0.25-1.5 mM CoC12. Enzyme activity was optimal in the
range
of 0.5 mM to 1 mM for both MnC12 and CoCl2. Hereafter 0.6 mM MnC12 was
employed in reactions or 0.5 mM CoC12. Reduced activity for cleavage of the
modified
substrate was observed using Mg buffers. Overall, optimum activity was
observed
using Mn buffers for cleavage of the di-fluoro (fNfN) substrates whereas Mg
buffers
were superior for cleavage of ribonucleotide (rN) substrates.
[0284] The ability of the RNase H2 enzymes to cleave at single or double 2'-
F
bases was unexpected. The Pyrococcus abyssi RNase H2 enzyme was next tested
for
the ability to cleave a greater variety of modified substrates using the same
methods
described above in this example. The modified strand of the substrate was
radiolabeled
as described above. Reactions were performed using 100 nM substrate and 480-
1000
mU of recombinant enzyme in Mn Cleavage Buffer (10 mM Tris pH 8.0, 50 mM NaC1,
0.6 mM MnC12, 10 jig/m1 BSA). Reactions were incubated at 70 C for 20 minutes.
Reaction products were separated using denaturing 7M urea, 15% polyacrylamide
gel
electrophoresis (PAGE) and visualized using a Packard CycloneTM Storage
Phosphor
System (phosphorimager). The relative intensity of each band was quantified
using the
manufacturer's image analysis software and results plotted as a fraction of
total
substrate cleaved are shown in Table 10.
Table 10: Cleavage of substrates containing 2'-modification by Pyrococcus
abyssi RNase H2
using increased amounts of enzyme
2'-Mod Oligo Sequence SEQ ID No. Cleavage
' -C TCGT GAGGT GAT ( fNfN) AGGAGATGGGAGGCG- 3 SEQ ID No.
fN-fN +++++
3'-GAGCACTCCACTA N N TCCTCTACCCTCCGC-5' 30
5'-CTCGTGAGGTGAT(EU+C)AGGAGATGGGAGGCG-3' SEQ ID No.
fU-LNA-C3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 31
5'-CTCGTGAGGTGAT(mUfC)AGGAGATGGGAGGCG-3' SEQ ID No.
mU-fC +++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 32
5'-CTCCTGAGGTGAT(mU+C)AGGAGATGGGAGGCG-3' SEQ ID No.
in U -LNA- C ++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 33
5'-CTCGTGAGGTGAT(mUmN)AGGAGATGGGAGGCG-3' SEQ ID No.
mU-mN ++
3'-GAGCACTCCACTA A N TCCTCTACCCTCCGC-5' 34
78
CA 02949315 2016-11-23
Table 10: Cleavage of substrates containing 2'-modification by Pyrococcus
abyssi RNasc H2
using increased amounts of enzyme
2'-Mod Oligo Sequence SEQ ID No. Cleavage
Amino-U-LN 5 -CTCGTGAGGTGAT ( aU+C) AGGAGATGGGAGGCG- 3 SEQ ID No.
A-C 3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 35
5'- CTCGTGAGGTGATG(fN)AGGAGATGGGAGGCG-3' SEQ ID No.
fN
3'- GAGCACTCCACTAC N TCCTCTACCCTCCGC-5' 36
5'-CTCGTGAGGTGAT(mUaC)AGGAGATGGGAGGCG-3' SEQ ID No.
mU-Amino-C +/-
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 37
LNA -T- fC 5'-CTCGTGAGGTGAT(+TfC)AGGAGATGGGAGGCG-3' SEQ ID No.
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 38
5'-
A U CTCGTGAGGTGATG(aU)AGGAGATGGGAGGCG-3' SEQ ID No.
mino-
3'- GAGCACTCCACTAC A TCCTCTACCCTCCGC-5' 39
LNA-T- 5'-
CTCGTGAGGTGAT(+T+C)AGGAGATGGGAGGCG-3 SEQ ID No.
[NA-C 3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 40
5'-CTCGTGAGGTGAT(fUmC)AGGAGATGGGAGGCG-3' SEQ ID No.
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 41
5'-CTCGTGAGGTGAT(+TmC)AGGAGATGGGAGGCG-3' SEQ ID No.
LNA-T-mC
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' 42
Uppercase letters = DNA; fN = 2'-F bases, +N = LNA bases, mN = 2'0Me bases, aN
= 2'-amino
bases
Use of "N" base indicates that every possible base (A, G, C, U/T) was tested
with the appropriate
perfect match complement. Efficiency of cleavage was rated from "+++++" (100%
cleavage) to
"-" (no cleavage). The mUmN substrates did not cleave equally well and the
"++" rating applies
to the best cleaving dinucleotide pair, mUmU. The rank order of cleavage for
this substrate
design was mUmU > mUmA > mUmC > mUmG.
102851 It is clear from the above results that many different 2'-
modifications can be
cleaved by RNase H2 enzymes that were not heretofore appreciated. Of the
2'-modified substrates, the di-fluoro compounds (those with 2 sequential 2'-
fluoro
bases) were most active. Additional substrates were tested, including some
with 3 or 4
sequential 2'-fluoro bases. No icrease in activity was seen when increasing
the
2'-fluoro content above 2 residues.
102861 A similar series of experiments was performed using lower amounts of
enzyme. The experiment below was conducted using an identical protocol except
that
148 U of recombinant Pyrococcus abyssi RNasc H2 was employed instead of the
480
mU previously employed (3000-fold less enzyme) and the buffer contained a
mixture
of divalent cations (3 mM MgC12 + 0.6 mM MnC12). Under these conditions, a
substrate containing a single ribonucleotide residue is completely cleaved
whereas
79
CA 02949315 2016-11-23
modified substrates are not. Results are shown in Table 11. RNase H2 is more
active in
cleaving substrates containing an RNA base than in cleaving the 2'-modified
bases.
Table 11. Cleavage of substrates containing 2'-modification by Pyrococcus
abyssi
RNase H2 using small amounts of enzyme
SEQ ID
2'-Mod Oligo Sequence No. Cleavage
5'-CTCGTGAGGTGATGnAGGAGATGGGAGGCG-3' SEQ ID
rN +++++
3'-GAGCACTCCACTACNTCCTCTACCCTCCGC-5' No. 43
5'-CTCGTGAGGTGAT(fUrC)AGGAGATGGGAGGCG-3' SEQ ID
fU-rC
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' No. 44
5'-CTCGTGAGGTGAT(rUfC)AGGAGATGGGAGGCG-3' SEQ ID
rU-fC ++++
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5' No. 45
5'-CTCGTGAGGTGAT(fNfN)AGGAGATGGGAGGCG-3' SEQ ID
fN-fN
3'-GAGCACTCCACTA N N TCCTCTACCCTCCGC-5' No. 30
5'- CTCGTGAGGTGATG(fN)AGGAGATGGGAGGCG-31 SEQ ID
fN
3'- GAGCACTCCACTAC N TCCTCTACCCTCCGC-5' No. 36
Uppercase letters = DNA; fN = 2'-F bases. Use of "N" base indicates that every
possible base (A, G, C, U/T) was tested with the appropriate perfect match
complement. Efficiency of cleavage was rated from "+++++" (100% cleavage) to "-
"
(no cleavage).
102871 Thus, Pyrococcus
abyssi RNase H2 can be used to cleave substrates which
do not contain any RNA bases but instead contain 2'-modified bases. Of the
compounds studied, di-fluoro (fNfN) containing substrates performed best. Use
of the
modified substrates generally requires increased amounts of enzyme, however
the
enzyme is catalytically very potent and it presents no difficulty to employ
sufficient
enzyme to achieve 100% cleavage of a di-fluoro substrate.
102881 The 2'-modified
substrate described in this example are not susceptible to
cleavage by typical RNasc enzymes. As such they can be employed in novel assay
formats where cleavage events are mediated by RNase H2 using substrates that
arc
completely resistant to cleavage by other RNase enzymes, particularly single
stranded
ribonucleases.
EXAMPLE 6 ¨ Base preferences for cleavage of the di-fluoro fNfN substrate
102891 The following
example demonstrates that all 16 possible 2'-fluoro
dinucleotides can be cleaved by RNase H2. Distinct base preferences are
observed.
CA 02949315 2016-11-23
102901 As shown in Table
12, the following 16 substrates were synthesized and
tested for efficiency of cleavage using the recombinant Pyrococcus abyssi
RNase H2
enzyme.
Table 12:
fNfN Sequence SEQ ID No.
5'-CTCGTGAGGTGATG(fAfA)GGAGATGGGAGGCG-3'
AASEQ ID No. 46
3'-GAGCACTCCACTAC T T CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fAfC)GGAGATGGGAGGCG-3'
ACSEQ ID No. 47
3'-GAGCACTCCACTAC T G CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fAfG)GGAGATGGGAGGCG-3'
AGSEQ ID No. 48
3'-GAGCACTCCACTAC T C CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fAfU)GGAGATGGGAGGCG-3'
AUSEQ ID No. 49
3'-GAGCACTCCACTAC T A CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fCfA)GGAGATGGGAGGCG-3'
CASEQ ID No. 50
3'-GAGCACTCCACTAC G T CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fCfC)GGAGATGGGAGGCG-3'
CCSEQ ID No. 51
3'-GAGCACTCCACTAC G G CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fCfG)GGAGATGGGAGGCG-3'
CGSEQ ID No. 52
3'-GAGCACTCCACTAC G C CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fCfU)GGAGATGGGAGGCG-3'
CUSEQ ID No. 29
3'-GAGCACTCCACTAC G A CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fGfA)GGAGATGGGAGGCG-3'
GASEQ ID No. 53
3'-GAGCACTCCACTAC C T CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fGfC)GGAGATGGGAGGCG-3'
GCSEQ ID No. 54
3'-GAGCACTCCACTAC C G CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fGfG)GGAGATGGGAGGCG-3'
GGSEQ ID No. 55
3'-GAGCACTCCACTAC C C CCTCTACCCTCCCC-5'
5'-CTCGTGAGGTGATG(fGfU)GGAGATGGGAGGCG-3'
GUSEQ ID No. 56
3'-GAGCACTCCACTAC C A CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fUfA)GGAGATGGGAGGCG-3'
UASEQ ID No. 57
3'-GAGCACTCCACTAC A T CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fUfC)GGAGATGGGAGGCG-3'
UCSEQ ID No. 58
3'-GAGCACTCCACTAC A G CCTCTACCCTCCGC-5'
5'-CTCGTGAGGTGATG(fUfG)GGAGATGGGAGGCG-3'
UGSEQ ID No. 59
3'-GAGCACTCCACTAC A C CCTCTACCCTCCGC-5'
81
CA 02949315 2016-11-23
Table 12:
fNfN Sequence SEQ ID No.
5'-CTCGTGAGGTGATG(fUfU)GGAGATGGGAGGCG-3'
UUSEQ ID No. 60
3'-GAGCACTCCACTAC A A CCTCTACCCTCCGC-5'
[0291] The modified strand
of each substrate was radiolabeled as described above.
Reactions were performed using 100 nM substrate with 25 mU of recombinant
enzyme
in Mn Cleavage Buffer (10 mM Tris pH 8.0, 50 mM NaCl, 0.6 mM MnC12, 10 pg/m1
BSA). Reactions were incubated at 70 C for 20 minutes. Reaction products were
separated using denaturing 7M urea, 15% polyacrylamide gel electrophoresis
(PAGE)
and visualized using a Packard CycloneTM Storage Phosphor System
(phosphorimager).
The relative intensity of each band was quantified, and results plotted as a
fraction of
total substrate cleaved are shown in Figure 14. The enzyme amount was titrated
so that
the most active substrate cleaved at 90-95% without having excess enzyme
present so
that accurate assessment could be made of relative cleavage efficiency for
less active
substrates.
102921 All 16 dinucleotidc
fININ pairs were cleaved by RNase H2, however clear
substrate preferences were observed. In general, substrates having the
sequence fNITI
performed worse, indicating that placement of a fU base at the 3'-position of
the
dinucleotide pair was unfavorable. The least active substrate was fUfU, which
showed
10% cleavage under conditions that resulted in >90% cleavage of fAfC or fAfG
substrates.
[0293] Using greater
amounts of enzyme, the relative differences of cleavage
efficiency between substrates is minor and 100% cleavage can readily be
achieved for
all substrates studied here.
EXAMPLE 7 ¨ Optimization of 3'- and 5'- base lengths for cleavage of rN and
fNfN substrates
[0294] The following
example shows the optimization of the placement of the
cleavable domain relative to the 3' and 5' ends of a primer or probe sequence.
In the
prior examples, the substrates all had 14 or 15 DNA bases on both the 5'- and
3'-sides
flanking the cleavable domain. For use in designing cleavable probes and
primers, it
82
CA 02949315 2016-11-23
may at times be beneficial to make these flanking sequences as short as
possible, in
order to control Tm (hybridization temperature) or to improve specificity of
priming
reactions. It is therefore important to define the minimum length of duplex
needed to
obtain efficient enzymatic cleavage.
[0295] In this
experiment, the synthetic substrate duplexes shown in Table 13 were
made having a single rC cleavable base, a fixed domain of 25 DNA bases 5'-
flanking
the ribonucleotide and a variable number of bases on the 3'-side.
Table 13:
3'-End Sequence (rC) SEQ ID No.
3' D1
5' -CTGAGCTTCATGCCTTTACTGTCCTeT-3'
-
3' -GACTCGAAGTACGGAAATGACAGGACA-5' SEQ ID No. 61
5' -CTGAGCTTCATGCCTTTACTGTCCTcTC-3'
3 ' -D2 SEQ ID No. 62
3' -GACTCGAAGTACGGAAATGACAGGACAG-5'
5'-CTGAGCTTCATGCCTTTACTGTCCTcTCC-3'
3'-D3 SEQ ID No. 63
3' -GACTCGAAGTACGGAAATGACAGGACAGG-5'
5' -CTGAGCTTCATGCCTTTACTGTCCTcTCCTT-3'
3'-D5 SEQ ID No. 64
3' -GACTCGAAGTACGGAAATGACAGGACAGGAA-5'
5' -CTGAGCTTCATGCCTTTACTGTCCTcTCCTTC-3'
3'-D6 SEQ ID No. 65
3' -GACTCGAAGTACGGAAATGACAGGACAGGAAG-5'
10296] The modified
strand of each substrate was radiolabeled as described above
Reactions were performed using 100 nM substrate with 100 itU of recombinant
enzyme
in Mg Cleavage Buffer (10 mM Tris pH 8.0, 50 mM NaCI, 4 mM MgC12, 10 ig/m1
BSA). Reactions were incubated at 70 C for 20 minutes. Reaction products were
separated using denaturing 7M urea, 15% polyacrylamide gel electrophoresis
(PAGE)
and visualized using a Packard CycloneTM Storage Phosphor System
(phosphorimager).
The relative intensity of each band was quantified, and results plotted as a
fraction of
total substrate cleaved are shown in Figure 15. Maximal cleavage occurred with
4-5
DNA bases flanking the ribonucleotide on the 3 '-side.
[0297] In the next
experiment, the synthetic substrate duplexes shown in Table 14
were made having a single rU cleavable base with a fixed domain of 25 base-
pairs
flanking the ribonucleotide on the 3'side and 2-14 base-pairs on the 5'-side.
A
83
CA 02949315 2016-11-23
minimum of 5 unpaired bases (dangling ends) were left on the unmodified
complement
to simulate hybridization to a long nucleic acid sample.
Table 14:
5'-End Sequence (rU) SEQ ID No.
5'-CuCCTGAGCTTCATGCCTTTACTGTCC-3'
SEQ ID No.
5'-D1 3' -ACGTAGAAT GGACAGAAGGAGGAC T C GAAGTACGGAAAT GACAGGACG TA- 5
66
5'-CCuCCTGAGCTTCATGCCTTTACTGTCC-3'
5'-D2 3'
SEQ ID No.
-ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA- 5
67
' -TCCuCCTGAGCTTCATGCCTT TACTGTCC - 3 '
SEQ ID No.
5 ' -D3 3' -ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA- 5
68
5 ' -TTCCuCCTGAGCTTCATGCCTT TACTGTCC- 3 '
SEQ ID No.
5'-D4 3' -ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA- 5
69
5'-CTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3'
SEQ ID No.
5'-D5 3' -ACG TAGAAT GGACAGAAGGAGGAC T CGAAGTACGGAAAT GACAGGACGTA- 5
5'-TCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3'
SEQ ID No.
5'-D6 3' -AC G TAGAAT GGACAGAAGGAGGAC T CGAAGTACGGAAAT GACAGGACGTA- 5
71
5'-TGTCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3'
5'-D8 3' -AC G TAGAAT GGACAGAAGGAGGAC T C GAAG TACGGAAAT GACAGGACGTA- 5 SEQ ID
No.
72
5' -CCTGTCTTCCuCCTGAGCTTCATGCCTTTACTGTCC- 3 ' SEQ ID No.
5 ' -D10 3' -ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA- 5
73
5'-TACCTGTCTTCCuCCTGAGCTTCATGCCTTTACTGTCC-3'
SEQ ID No.
5 ' -D12 3 ' -ACGTAGAATGGACAGAAGGAGGACTCGAAGTACGGAAATGACAGGACGTA- 5
74
5 ' -CT TACCTGTC T TCCuCCTGAGCTTCATGCCTTTACTGTCC- 3 '
SEQ ID No.
5 ' -D14 3 ' -ACGTAGAAT GGACAGAAGGAGGAC T C GAAGTACGGAAAT GACAGGACGTA- 5
102981 The modified strand of each substrate was radiolabeled as previously
described. Reactions were performed using 100 nM substrate with 123 j.iU of
recombinant enzyme in a mixed buffer containing both Mg and Mn cations (10 mM
Tris pH 8.0, 50 mM NaCl, 0.6 mM MnC12, 3 mM MgCl2, 10 g/m1 BSA). Reactions
were incubated at 70 C for 20 minutes. Reaction products were separated using
denaturing 7M urea, 15% polyaerylamide gel electrophoresis (PAGE) and
visualized
using a Packard CycloneTm Storage Phosphor System (phosphorimager). The
relative
intensity of each band was quantified and the results plotted as a fraction of
total
substrate cleaved in Figure 16. Little cleavage was seen with the short
substrates.
Activity increased with length of the 5'-DNA domain until maximum cleavage was
obtained at around 10-12 bases of duplex flanking the rU base on the 5'-side.
84
CA 02949315 2016-11-23
[0299] Similar experiments were done to determine the optimal length of the
3'-
DNA domain needed for cleavage of di-fluoro (fNfN) substrates. The duplexes
shown
in Table 15 were synthesized and tested to functionally define the length of
DNA bases
needed at the 3'-end of a fUfC di-fluoro substrate. A fixed domain of 22 base
pairs was
positioned at the 5'-end and the 3'-domain was varied from 2-14 bases.
Table 15:
3'-End Sequence (fUfC) SEQ ID No.
3' D2
5'-CTGAGCTTCATGCCTTTACTGT(fUfC)CC-SpC3-3' SEQ ID No.
-
3'-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 76
3' D4 5'-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCG-SpC3-3' SEQ ID No.
-
3'-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 77
3' D5
5'-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGA-SpC3-3' SEQ ID No.
-
3'-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 78
3' D6 5f-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGAC-SpC3-3' SEQ ID No.
-
3'-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 79
3' D8
5'-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACAC-SpC3-3' SEQ ID No.
-
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 80
3' D 10 5'-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACACAC-SpC3-3' SEQ ID No.
-
3f-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 81
3' D12 5'-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACACACAG-SpC3-3' SEQ ID No.
-
3'-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 82
3' D14
5'-CTGAGCTTCATGCCTTTACTGT(fUfC)CCCGACACACAGCT-SpC3-3' SEQ ID No.
-
3'-GACTCGAAGTACGGAAATGACA A G GGGCTGTGTGTCGAG-5' 83
[0300] The modified strand of each substrate was radiolabeled as above.
Reactions
were performed using 100 nM substrate with 37 mU of recombinant enzyme in a
mixed
buffer containing both Mg and Mn cations (10 mM Tris pH 8.0, 50 mM NaC1, 0.6
mM
MnC12, 3 mM MgC12, 10 jig/ml BSA). Reactions were incubated at 70 C for 20
minutes. Reaction products were separated using denaturing 7M urea, 15%
polyacrylamide gel electrophoresis (PAGE) and visualized using a Packard
CycloneIm
Storage Phosphor System (phosphorimager). The relative intensity of each band
was
quantified and the results plotted as a fraction of total substrate cleaved in
Figure 17.
No cleavage was seen with the substrate having 2 DNA bases on the 3'-side of
the
cleavable domain. Cleavage was seen with 4 DNA bases and steadily increased
until
maximal cleavage was obtained when 8-10 DNA bases were present on the 3'-side
of
the fUfC cleavage domain. Interestingly, the optimal length of DNA bases on
the
3'-side of the cleavage domain is longer for the di-fluoro substrates (8-10
bases)
compared with the single ribonucleotide substrates (4-5 bases).
CA 02949315 2016-11-23
[0301] In summary, for
ribonucleotide containing substrates, maximal cleavage
activity is seen when at least 4-5 DNA residues are positioned on the 3'-side
and 10-12
DNA residues are positioned on the 5'-side of the cleavable domain. For di-
fluoro
substrates, maximal cleavage activity is seen when at least 8-10 DNA residues
are
positioned on the 3'-side of the cleavable domain; from prior examples it is
clear that
activity is high when 14-15 DNA residues are positioned on the 5'-side of the
cleavable
domain.
EXAMPLE 8 ¨ Application to DNA primers: primer extension assay format and
potential utility in DNA sequencing
103021 The examples
above characterized the ability of a thermostable RNase H2
enzyme to cleave a duplex nucleic acid at a single internal ribonucleotide or
at a
2'-fluoro dinucleotide. Example 7
establishes parameters for designing short
oligonucleotides which will be effective substrates in this cleavage reaction.
These
features can be combined to make cleavable primers that function in primer
extension
assays, such as DNA sequencing, or PCR. A single stranded oligonucleotide is
not a
substrate for the cleavage reaction, so a modified oligonucleotide primer will
be
functionally "inert" until it hybridizes to a target sequence. If a cleavable
domain is
incorporated into an otherwise unmodified oligonucleotide, this
oligonucleotide could
function to prime PCR and will result in an end product wherein a sizable
portion of the
primer domain could be cleaved from the final PCR product, resulting in
sterilization of
the reaction (lacking the priming site, the product will no longer be a
template for PCR
using the original primer set). If the cleavable domain is incorporated into
an
oligonucleotide which is blocked at the 3'-end, then this primer will not be
active in
PCR until cleavage has occurred. Cleavage will "activate" the blocked primer.
As
such, this format can confer a "hot start" to a PCR reaction, as no DNA
synthesis can
occur prior to the cleavage event. Example 4 showed that this cleavage event
is very
inefficient with Pyrococcus abysii RNasc H2 until elevated temperatures are
attained.
Additionally, the linkage between the cleavage reaction and primer extension
confer
added specificity to the assay, since both steps requireenzymatic recognition
of the
duplex formed when the primer hybridizes to the template. A schematic of this
reaction
is shown in Figure 18. Note that this schema applies to both simple primer
extension
86
CA 02949315 2016-11-23
reactions as well as PCR. It can also be exploited in other kinds of enzymatic
assays
such as ligation reactions.
[0303] The following example demonstrates the use of an RNase H2 cleavable
primer for DNA sequencing. The most common method of DNA sequencing in use
today involves sequential DNA synthesis reactions (primer extension reactions)
done in
the presence of didcoxy terminator nucleotides. The reaction is done in a
thermal
cycling format where multiple cycles of primer extension are performed and
product
accumulates in a linear fashion.
[0304] DNA sequencing was done using the Big DyeTM Terminator V3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA). The following primers
were
used:
M13(-27)
[0305] SEQ ID No. 84
5' -CAGGAAACAGCTATGAC-3'
M13(-27)-rC
[0306] SEQ ID No. 85
5' -CAGGAAACAGCTATGACcATGA-SpC3-3'
103071 As before, DNA bases are indicated in upper case, RNA bases are
indicated
in lower case, and SpC3 is a spacer C3 blocking group placed at the 3'-end of
the
oligonucleotide. The blocked cleavable primer contains 17 DNA bases on the 5'-
side
of the ribonucteotide and 4 DNA bases on the 3'-side of the ribonucleotide (17-
1-4
design) and so conforms to the optimized design rules established in Example
7.
[0308] Sequencing reactions were set up in 20 1 volume comprising 0.75X
ABI
Reaction buffer, 160 nM primer, 0.5X Big Dye Terminators and 230 ng plasmid
DNA
template. Optionally, 4 mM additional MgC12 was supplemented into the
reaction, with
or without 14, 1.4, or 0.14 mU of recombinant Pyrococcus abyssi RNase H2. The
following cycle sequencing program was employed: 96 C for 30 seconds followed
by
25 cycles of [96 C for 5 seconds, 50 C for 10 seconds, 55 C for 4 minutes].
The DNA
sequencing reactions were run on an Applied Biosystems model 3130x1 Genetic
87
CA 02949315 2016-11-23
Analyzer. The resulting sequencing traces were examined for quality and read
length.
Results are summarized in Table 16 below.
Table 16. Results of cycle sequencing using a rC blocked cleavable primer
Read length in ABI Read length in ABI
Primer RNase H2
Buffer Buffer + 4 mM MgC12
0 >800 ¨500
M13(-27) 0.14 mU >800 >800
SEQ ID No. 84 1.4 mU >800 >800
14 mU >800 >800
0 0 0
M13(-27)-rC 0.14 mU 0 0
SEQ ID No. 85 1.4 mU 0 ¨300
14 mU ¨300 >800
[03091 Control reactions using an unmodified primer resulted in high
quality DNA
sequence traces with usable read lengths slightly exceeding 800 bases. The
addition of
RNase H2 enzyme to these reactions did not compromise reaction quality. The
manufacturer (Applied Biosystems) does not disclose the cation content of the
buffer
provided in the sequencing kits, so actual reaction conditions are not
certain.
Supplementation of the reactions with an additional 4 mM MgCl2 had no effect.
The rC
blocked cleavable primer did not support DNA sequencing without the addition
of
RNase H2. With the addition of RNase H2, high quality sequencing reactions
were
obtained using 14 mU of enzyme in the 20 i.t1 reaction. Use of lower amounts
enzyme
resulted in lower quality reactions or no functional reaction at all.
Supplementing
magnesium content of the reaction buffer was necessary to obtain cleavage and
primer
extension reactions using the blocked primers. The amount of enzyme employed
here
is 100-fold higher than is needed to achieve 100% cleavage of a rN substrate
under
optimal conditions (70 C, 20 minute incubation). In the cycle sequencing
reactions
performed herein, primer annealing was run at 50 C and extension reactions
were run at
55 C for 10 seconds and 4 minutes, respectively. These lower temperatures are
suboptimal for Pyrococcus abyssi RNase H2 (see Example 4 above). Performing
the
88
CA 02949315 2016-11-23
cycle sequencing reaction at higher temperatures will require less enzyme but
is not
necessary.
103101 This example demonstrates that blocked primers containing an
internal
cleavage site for RNase H2 can be used with primer-extension based sequencing
methods, such as dideoxy (Sanger) sequencing, and are compatible with use of
existing
high throughput fluorescent sequencing protocols. Use of blocked primers and
the
method of the present invention can confer added specificity to the sequencing
reaction,
thus permitting sequencing to be performed for more cycles and on highly
complex
nucleic acid samples that work poorly with unmodified primers.
EXAMPLE 9 ¨ Application to DNA primers: rN primers in PCR and quantitative
real-time PCR
103111 Example 8 demonstrated that RNase H2 could be used to cleave a
blocked
primer and that this system could be linked to DNA synthesis and primer
extension
reactions, including DNA sequencing. The following example demonstrates the
utility
of this method in PCR. The first system demonstrates use in an end point PCR
format
and the second system demonstrates use in a quantitative real-time PCR format.
[0312] The primers shown in Table 17, were made for use in a synthetic end-
point
PCR assay. The Syn-For and Syn-Rev primers are unmodified control primers
specific
for an artificial amplicon (a synthetic oligonucleotide template). The Syn-For
primer is
paired with the unmodified control Syn-Rev primer or the different modified
Syn-Rev
primers. A set of modified Syn-Rev primers were made which contain a single rU
(cleavable) base followed by 2-6 DNA bases, all ending with a didcoxy-C
residue
(ddC). The ddC residue functions as a blocking group that prevents primer
function.
The ddC blocking group is removed with cleavage of the primer at the rU base
by the
action of RNase H2 (the unblocking step, shown in Figure 18). The synthetic
template
is a 103-base long oligonucleotide, shown below (SEQ ID No. 93). Primer
binding
sites are underlined.
89
CA 02949315 2016-11-23
Table 17:
Name Sequence SEQ ID No.
Syn-For 5' -AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 86
Syn-Rev 5' -CTGAGCTTCATGCCTTTACTGT-3' SEQ ID No. 87
Syn-Rev-rU-2D 5' -CTGAGCTTCA1GCCTTTACTGTuCC-ddC-3' SEQ ID No. 88
Syn-Rev-rU-3D 5' -CTGAGCTTCATGCCTTTACTGTuCCC-ddC-3f SEQ ID No. 89
Syn-Rev-rU-4D 5' -CTGAGCTTCATGCCTTTACTCTuCCCC-ddC-3f SEQ ID No. 90
Syn-Rev-rU-5D 5' -CTGAGCTTCATGCCTTTACTGTuCCCCG-ddC-3' SEQ ID No. 91
Syn-Rev-rU-6D 5' -CTGAGCTTCATGCCTTTACTGTuCCCCGA-ddC-3f SEQ ID No. 92
DNA bases are shown in uppercase. RNA bases arc shown in lowercase. ddC
indicates a
dideoxy-C residue which functions as a blocking group.
Synthetic template
103131 SEQ ID No. 93
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
103141 PCR reactions were performed in 20 ul volume using 200 nM primers, 2
ng
template, 200 )..tM of each dNTP (800 M total), 1 unit of Immolase (a
thermostable
DNA polymerase, Bioline), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM MgC12.
Reactions were run either with or without 100 uU of Pyrococcus abyssi RNase
H2.
Reactions were started with a soak at 95 C for 5 minutes followed by 35 cycles
of [95 C
for 10 seconds, 60 C for 30 seconds, and 72 C for 1 second]. Reaction products
were
separated on a 10% non-denaturing polyacrylamide gel and visualized using
GelStar
staining. Results are shown in Figure 19. Unmodified control primers produced
a
strong band of the correct size. 3'-end blocked rU primers did not produce any
products in the absence of RNase H2. In the presence of RNase H2, blocked
primers
produced a strong band of the correct size using the D4, D5, and D6 primers.
No signal
was seen using the D2 or D3 primers. This example demonstrates that blocked
primers
can be used in PCR reactions using the method of the present invention.
Further, this
example is consistent with results obtained using cleavage of preformed duplex
CA 02949315 2016-11-23
substrates in Example 7, where the presence of 4-5 3'-DNA bases were found to
be
optimal for cleavage of rN containing primers.
103151 The same synthetic PCR amplicon assay system described above was
next
tested in a quantitative real-time PCR assay using SYBle Green detection.
Reactions
were done in 384 well format using a Roche Lightcycler 480 platform.
Reactions
comprised lx BIO-RAD iQTm SYBRR' Green Supermix (BIO-RAD, Hercules, CA),
200 nM of each primer (for + rev), 2 x 106 copies of synthetic template
oligonucleotide
(SEQ ID No. 93), and 5 mU of Pyrococcus abyssi RNase H2 in 10 gl volume.
Thermal
cycling parameters included an initial 5 minutes soak at 95 C and then 45
cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds]. All
reactions were run in triplicate and reactions employed the same unmodified
For primer
(SEQ ID No. 86). The Rev primer was varied between the unmodified and 2-6D
modified primers (SEQ ID Nos. 87-92). Cp values, the PCR cycle number where a
positive reaction is first detected, in these experiments are shown in Table
18 below.
The Cps were essentially identical for control reactions done using unmodified
For +
Rev primers and the coupled cleavage PCR reactions performed using the D4, D5,
or
D6 blocked primers in the presence of RNase H2. In the absence of RNase H2, no
positive signal was detected using the blocked primers. As was seen in the end
point
assay, performance was reduced for the primers having shorter 3'-DNA domains
(D2
or D3).
Table 18. Cp values for SYBRI. Green qPCR reactions using
cleavable blocked primers in a synthetic amplicon system with
RNase H2 present
Reverse Primer SEQ ID No. Cp Value
Syn-Rev (Control) SEQ ID No. 87 17.7
Syn-Rev-rU-2D SEQ ID No. 88 23.4
Syn-Rev-rU-3D SEQ ID No. 89 23.0
Syn-Rev-rU-4D SEQ ID No. 90 16.8
Syn-Rev-rU-5D SEQ ID No. 91 16.6
Syn-Rev-rU-6D SEQ ID No. 92 16.9
All reactions used the same unmodified For primer, SEQ ID No.
86
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CA 02949315 2016-11-23
[0316] The following
example demonstrates use of RNase H2 cleavage using rN
blocked primers (both For and Rev) in a quantitative real-time PCR assay
format using
an endogenous human gene target and HeLa cell cDNA as template. The primers
shown in Table 19 specific for the human HRAS gene (NM 176795) were designed
and synthesized. In this case a C3 spacer was usc das the blocking group.
Table 19:
SEQ ID
Name Sequence
No.
SEQ
HRAS-618-For 5f-ACCTCGGCCAAGACCC-3' ID No.
94
S
HRAS-916-Rev 5'-CCTTCCTTCCTTCCTTGCTTCC-3' EQ ID No.
HRAS-6 I 8-For-rG- SEQ ID No.
5f-ACCTCGGCCAAGACCCgGCAG-SpC3-3'
D4 96
HRAS-916-Rev-rG- SEQ ID No.
5'-CCTTCCTTCCTTCCTTGCTTCCgTCCT-SpC3-3'
D4 97
Uppercase represents DNA bases, lowercase represents RNA bases. SpC3 is a
spacer
C3 placed as a blocking group on the 3 '-end.
[0317] These primers
define a 340 bp amplicon within the HRAS gene as shown
below. Primer binding sites are underlined.
HRAS assay amplicon
[0318] SEQ ID No. 98
ACCTCGGCCAAGACCCGGCAGGGCAGCCGCTCTGGCTCTAGCTCCAGCTCCGGGACCCTCTGGGACCCC
CCGGGACCCATGTGACCCAGCGGCCCCTCGCGCTGGAGTGGAGGATGCCTTCTACACGTTGGTGCGTGA
GATCCGGCAGCACAAGCTGCGGAAGCTGAACCCTCCTGATGAGAGTGGCCCCGGCTGCATGAGCTGCAA
GTGTGTGCTCTCCTGACGCAGCACAAGCTCAGGACATGGAGGTGCCGGATGCAGGAAGGAGGTGCAGAC
GGAAGGAGGAGGAAGGAAGGACGGAAGCAAGGAAGGAAGGAAGG
[0319] Reactions were
performed in 10 .1.1 volume in 384 well format using a
Roche Lightcycler( 480 platform. Reactions comprised lx BIO-RAD iQTM SYBR
Green Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at
25U/ml, 3 mM MgC12, 200 nM of each primer (for + rev), 2 ng cDNA (made from
HeLa cell total RNA), with or without 5 mU ofPyrococcus abyssi RNase H2.
Thermal
cycling parameters included an initial 5 minutes soak at 95 C and then 50
cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds]. All
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CA 02949315 2016-11-23
reactions were run in triplicate. Using unmodified primers, the crossing point
(Cp)
occurred at cycle 27. In the absence of RNase H2, reactions done with blocked
primers
did not support PCR and no fluorescence signal was detected during the 50
cycle
reaction. In the presence of RNase H2, reactions done with blocked primers
produced
detectable signal at cycle 27.4, essentially identical to the control
unblocked primers.
Real time PCR fluorescence plots are shown in Figure 20.
[0320] The following example demonstrates use of RNase H2 cleavage using rN
blocked primers in a quantitative real-time PCR assay format using another
endogenous human gene target and HeLa cell cDNA as the template. The primers
specific for the human ETS2 gene (NM_005239) shown in Table 20 were designed
and
synthesized.
Table 20:
Name Sequence SEQ ID No.
ETS2-300-For 5'-CCCTGTTTGCTGTTTTTCCTTCTC-3' SEQ ID No. 99
ETS2-463-Rev 5f-CGCCGCTGTTCCTTTTTGAAG-3' SEQ ID No. 100
ETS2-300-For-rU-D4 5' -CCCTGTTTGCTGTTTTTCCTTCTCuAAAT-SpC3-3' SEQ ID No. 101
ETS2-463-Rev-rC-D4 5' -CGCCGCTGTTCCTTTTTGAAGcCACT-SpC3-3' SEQ ID No. 102
Uppercase represents DNA bases, lowercase represents RNA bases. SpC3 is a
spacer C3 placed
as a blocking group on the 3'-end
[0321] These primers define a 184 bp amplicon within the ETS2 gene as shown
below. Primer binding sites are underlined.
ETS2 assay amplicon
103221 SEQ ID No. 103
CCCTGTTTGCTGTTTTTCCTTCTCTAAATGAAGAGCAAACACTGCAAGAAGTGCCAACAGGCTTGGATT
CCATTTCTCATGACTCCGCCAACTGTGAATTGCCTTTGTTAACCCCGTGCAGCAAGGCTGTGATGAGTC
AAGCCTTAAAAGCTACCTTCAGTGGCTTCAAAAAGGAACAGCGGCG
10323] Reactions were performed in 10 ul volume in 384 well format using a
Roche Lightcycler 480 platform. Reactions comprised lx BIO-RAD jQTMSYBIe
93
CA 02949315 2016-11-23
Green Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at
25U/ml, 3 mM MgC12, 200 nM of each primer (for + rev), 2 ng cDNA (made from
HeLa cell total RNA), with or without 5 mU ofPyrococcus abyssi RNase H2.
Thermal
cycling parameters included an initial 5 minutes soak at 95 C and then 50
cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds]. All
reactions were run in triplicate. Using unblocked primers, the Cp occurred at
cycle 25.7.
In the absence of RNase H2, reactions done with blocked primers did not
support PCR
and no fluorescence signal was detected out to 50 cycles. In the presence of
RNase H2,
reactions done with blocked primers produced detectable signal at cycle 31.7,
a delay of
6 cycles from the unmodified control primers. Reactions done using one blocked
primer (unmodified For + blocked Rev or blocked For + unmodified Rev) showed
intermediate Cp values. Real time PCR fluorescence plots are shown in Figure
21.
103241 Using the present reaction conditions, the HRAS assay performed
identically using unmodified vs. blocked primers. However, the ETS2 assay
showed a
delay between unmodified vs. blocked primers. In the setting of a PCR reaction
where
rapid thermal cycling occurs, primer hybridization and cleavage kinetics play
a
significant role in the efficiency of the overall reaction for reactions which
employ the
blocked primers. DNA synthesis is linked to the unblocking event, and
unblocking
requires hybridization, binding of RNase H2, and substrate cleavage before
primers
become activated and are capable of priming DNA synthesis. It should be
possible to
increase the amount of cleaved primer produced each cycle by either increasing
the
amount of RNase H2 enzyme present or by increasing the anneal time of the
reaction.
DNA synthesis occurs at the anneal temperature (60 C) nearly as well as at the
extension temperature (72 C) used in the above examples. However, unblocking
can
only take place during the duration of the anneal step (60 C) and not during
the extend
step (72 C) due to the Tm of the primers employed which only permit formation
of a
double-stranded substrate for RNase H2 during the anneal step but not at 72 C
(where
the primers only exist in single-stranded form).
103251 PCR cycle parameters were changed to a 2 step reaction with
anneaUextend
as a single event done at 60 C and the duration of the anneal/extend step was
varied to
see if changing these reaction parameters could allow the blocked ETS2 primers
to
perform with similar efficiency as the unmodified control primers. Reactions
were
94
CA 02949315 2016-11-23
done in 10 I volume in 384 well format using a Roche Lightcycler 480
platform.
Reactions comprised lx BIO-RAD iQTM SYBR Green Supermix (BIO-RAD,
Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3 mM MgC12, 200 nM of
each primer (for + rev), 2 ng cDNA (made from HeLa cell total RNA), with or
without
mU of Pyrococcu.s' abyksi RNasc H2. Thermal cycling parameters included an
initial
5 minutes soak at 95 C and then 45 cycles were performed of [95 C for 10
seconds +
60 C for 20-120 seconds]. All reactions were run in triplicate. The
differences
between the Cp values obtained for the blocked primers and the unmodified
control
primers (ACp) are summarized in Table 21 below.
Table 21. ACp values for SYBR Green qPCR ETS2 reactions
comparing unmodified and cleavable blocked primers
Combined time ACp Value
at 60 C (anneal/extend)
20 seconds 6.1
60 seconds 1.2
90 seconds 0.6
120 seconds 0.4
103261 Minor adjustment of the cycling parameters and increasing the
duration of
the 60 C anneal step from 20 seconds to 1-2 minutes led to uniform performance
between the blocked-cleavable primers and the control unmodified primers.
Similar
experiments were performed keeping the cycling parameters fixed and increasing
enzyme. As predicted, it was possible to improve performance of the blocked
primers
using higher amounts of enzyme. Doubling the amount of enzyme employed to 10
mU
RNase H resulted in minimal difference between control unblocked and blocked
cleaveable primers when using a 30 second anneal step at 60 C.
[0327] The above example demonstrates that blocked primers containing a
single
ribonucleotide residue of the optimized design taught in Example 7 can be used
with
RNase H2 in quantitative real-time PCR assays.
CA 02949315 2016-11-23
EXAMPLE 10¨ Application to DNA primers: fNfN primers in PCR and
quantitative real-time PCR
103281 Example 9 above demonstrated utility of RNasc H2 mediated cleavage
for
use of rN blocked primers in end point and quantitative real time PCR assays.
The
present example demonstrates utility using fMN blocked primers in quantitative
real
time PCR assays.
103291 Since cleavage of the di-fluoro substrate by RNasc H2 results in a
species
having a 3'-OH end, this product should also be able to support PCR reactions
using the
same reaction format as described in Example 9, assuming that primers bearing
a single
2'-F base (fN) are capable of priming DNA synthesis. Cleavage of a di-fluoro
substrate
proceeds best in the presence of manganese cations, whereas PCR reactions
generally
are performed in the presence of magnesium cations. PCR reactions using
unmodified
primers were tested using standard qPCR buffer containing 3 mM MgC12 and a
modified buffer containing 3 mM MgCl2 + 0.6 mM MnC12. Reaction performance was
identical and the presence of this low amount of manganese did not adversely
affect the
quantitative nature of the reaction.
103301 The ability of a terminal 3'-fN primer to function in PCR was
investigated
using the synthetic PCR amplicon system described in example 9. The following
primers shown in Table 22 were tested:
Table 22:
Name Sequence SEQ ID No.
Syn-For 5'-AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 86
Syn-Rev 5'-CTGAGCTTCATGCCTTTACTGT-3' SEQ ID No. 87
Syn-Rev-fU 5'-CTGAGCTTCATGCCTTTACTGT(fU)-3' SEQ ID No. 104
DNA bases are shown in uppercase. 2'-fluoro bases are indicated as fN.
96
CA 02949315 2016-11-23
Synthetic template
103311 SEQ ID No. 93
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0332] Reactions were done in 10 ttl volume in 384 well format using a
Roche
Lightcyclerg 480 platform. Reactions comprised lx BIO-RAD jQTM SYBR Green
Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3
mM MgC12, 0.6 mM MnC12, 200 nM of each primer (for + rev), 2 x 106 copies of
synthetic oligonucleotide target, with or without 1.75 U of Pyrococcus- abyssi
RNase
H2. Thermal cycling parameters included an initial 5 minutes soak at 95 C and
then 30
cycles were performed of [95 C for 10 seconds + 60 C for 120 seconds + 72 C
for 120
seconds]. All reactions were run in triplicate. Results are shown in Figure
22. In the
absence of RNase H2, the primer having a 2'-F base at the 3'-end supported PCR
with
identical efficiency compared with the unmodified primer. However, in the
presence of
RNase H2, the 2'-F modified primer showed a 3.5 Cp delay compared with the
unmodified primer. This results not from the inhibition of DNA synthesis by
RNase H2,
but from a low level of cleavage of the primer from the amplification product
by RNase
H2. Following DNA synthesis, incorporation of a fN-containing primer into the
newly
formed DNA product creates a potential substrate for RNase H2 (see example 5
above).
Cleavage at the 2'-F base will remove the priming site from this strand of the
amplicon,
effectively sterilizing this product so that any products made from it will be
incapable
of further priming events. It is this reaction sequence which occurs in
polynomial
amplification. Cleavage of substrates containing a single 2'-F residue is
relatively
inefficient, however, so only a modest decrease in PCR reaction efficiency is
seen.
Extending incubation at 72 C following PCR should result in total cleavage of
the
primer from the amplification product, completely blocking the ability of
further
amplification to occur and thereby sterilizing the product. This should be
useful in
cross-contamination control of PCR reactions.
103331 Given that the cleavage of a single 2'-F residue is inefficient, use
of lower
amounts of enzyme, or eliminating the 72 C elongation step permits cleavage
of a
difluoro blocked primer by RNase H2 without significantly cleaving the primer
extension reaction product containing a single 2' fluoro residue.
Alternatively, it
97
CA 02949315 2016-11-23
should be possible to block this cleavage event by selective placement of a
phosphorothioate modification between the terminal 2'-F residue and the
adjacent
DNA base.
[0334] The ability of a di-fluoro blocked primer to support ciPCR was
demonstrated
using the primers shown in Table 23, in the synthetic oligonucleotide amplicon
system,
described in Example 9 above.
Table 23:
Name Sequence SEQ ID No.
Syn-For 5'-AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 86
Syn-Rev-fU 5'-CTGAGCTTCATGCCTTTACTGT(fU)-3' SEQ ID No. 104
Syn-Rev-fUfC-D 5' -CTGAGCTTCATGCCTTTACTGT (fUfC) CCCGACACA
S
C-SpC3-3' EQ ID No. 105
DNA bases are shown in uppercase. 2'-F bases are indicated as fN. SpC3
indicates a spacer
C3 group employed to block the 3'-end
Synthetic template
[0335] SEQ ID No. 93
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
103361 Reactions were done in 10 !Al volume in 384 well format using a
Roche
Lightcycler 480 platform. Reactions comprised lx BIO-RAD iQTm SYBle Green
Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA polymerase at 25U/ml, 3
mM MgC12, 0.6 mM MnC12, 200 nM of each primer (for + rev), 2 x 106 copies of
synthetic oligonucleotide target, with or without 1.75 U of Pyrococcus abyssi
RNasc
H2. Thermal cycling parameters included an initial 5 minutes soak at 95 C and
then 45
cycles were performed of [95 C for 10 seconds + 60 C for 120 seconds + 72 C
for 120
seconds]. All reactions were run in triplicate. The reactions run with the
control primer
having a single 2'-fluoro base at the 3'-end (which mimics the cleavage
product of the
fNfN blocked primer) had a Cp of 20. Reactions run with the blocked fUfC
primer also
had a Cp of 20.
[0337] The amount of RNase H2 enzyme needed in the di-fluoro primer
cleavage
assay was next studied in more detail. Reactions were done in 10 j..11 volume
in 384 well
98
CA 02949315 2016-11-23
format using a Roche Lightcycler 480 platform. Reactions comprised lx BIO-RAD
iQIM SYBR Green Supermix (BIO-RAD, Hercules, CA) using the iTAQ DNA
polymerase at 25U/ml, 3 mM MgC12, 0.6 mM MnC12, 200 nM of each primer (for +
rev), 2 x 106 copies of synthetic oligonucleotide target. The same unmodified
Syn-For
primer was used in all reactions. Recombinant Pyrococcus abyssi RNasc H2 was
added from 0 to 600 mU per reaction. Thermal cycling parameters included an
initial 5
minutes soak at 95 C and then 45 cycles were performed of [95 C for 10 seconds
+
60 C for 120 seconds + 72 C for 120 seconds]. All reactions were run in
triplicate. Cp
values corresponding to the varying amounts of RNase H2 for each primer are
shown in
Table 24.
Table 24. Optimization of the amount of RNase H2 for ciPCR reactions using a
fUfC
blocked primer
Amount of RNase H2 used per reaction
Primer 600 mU 400 mU 200 mU 100 mU 0 mU
Syn-Rev 17.9 17.7 17.2 17.1 17.0
Syn-Rev-fU 25.6 23.2 19.9 18.5 17.0
Syn-Rev-111 24.6 22.9 21.3 21.9 ND
fC-D10
ND = not detected.
[0338] The optimal amount of RNase H2 is 200 mU (Cp = 21.3 shown in bold
and
underlined). At higher concentrations of RNase H2 PCR reaction is less
efficient, and
to a similar degree, with both the 3' fluoroU primer and the blocked difluoro
primer.
Presumably this is due to a low level of cleavage at the fU set within the PCR
product as
discussed above.
[0339] Generally about 200 mU of Pyrococcus abysii RNase H2 per 10 p1 is
the
optimal enzyme concentration for a coupled RNase H2-PCR with blocked primers
wherein the RNase H2 cleavage domain is two consecutive 2'-fluoronucleosides.
An
increase in Cp compared to standard unmodified DNA primers of between 2 and 6
cycles is typically observed. This small difference has no effect on assay
performance
because results are always compared to a standard curve of Cp vs. target copy
number
generated with the same primers as used to test unknown samples.
99
CA 02949315 2016-11-23
[0340] In conclusion, this example has demonstrated that blocked fNfN
primers
can support ciPCR reactions using RNase H2 cleavage with the methods of the
present
invention and defines optimal amounts of RNase H2 and cycling conditions to
employ.
EXAMPLE 11 ¨ Improved specificity using rN blocked primers in PCR reactions.
[0341] In theory, PCR has an almost unlimited potential for amplification
and a
PCR reaction should only be limited by consumption of reagents in the reaction
mix. In
actual practice, PCR reactions are typically limited to 40-45 cycles to help
preserve
specificity. The amplification power of PCR is enormous and, as cycle number
exceeds
40-45, it becomes increasingly common for mispriming events to give rise to
amplification of undesired products and false positive signals. This example
demonstrates how use of cleavable blocked primers with the methods of the
present
invention improves reaction specificity and permits use of a greater number of
PCR
cycles, thereby increasing the potential sensitivity of PCR.
[0342] In this example, we studied PCR reactions specific for 3 human genes
and
compared the specificity of each set of primer pairs in amplification using
human and
rat cDNA as the template. Traditional unmodified oligonucleotides were
compared
with the new cleavable blocked primers of the present invention. The following
primers, as shown in Table 25, were employed. DNA bases are shown in upper
case,
RNA bases in lower case, and the 3'-blocking group employed was a C3 spacer
(SpC3).
The gene targets studied were human ETS2, NM 005239 (rat homolog
NM 001107107), human HRAS, NM 176795 (rat homolog NM 001061671), and
human ACACA, NM 198834 (rat homolog NM 022193).
100
CA 02949315 2016-11-23
Table 25:
Gene Primer SEQ ID No. Sequence
ETS2 hETS2-For SEQ ID No. 106 CCCTGTTTGCTGTTTTTCCTTCTC
hETS2-For-rU SEQ ID No. 107 CCCTGTTTGCTGTTTTTCCTTCTCuAAAT-SpC3
hETS2-Rev SEQ ID No. 108 CGCCGCTGTTCCTTTTTGAAG
hETS2-Rev-rC SEQ ID No. 109 CGCCGCTGTTCCTTTTTGAAGcCACT-SpC3
HRAS hHRAS-For SEQ ID No. 110 ACCTCGGCCAAGACCC
hHRAS-For-rG SEQ ID No. 111 ACCTCGGCCAAGACCCgGCAG-SpC3
hHRAS-Rev SEQ ID No. 112 CCTTCCTTCCTTCCTTGCTTCC
hHRAS-Rev-rG SEQ ID No. 113 CCTTCCTTCCTTCCTTGCTTCCgTCCT-SpC3
ACACA hACACA-For SEQ ID No. 114 GCATTTCTTCCATCTCCCCCTC
hACACA-For-rU SEQ ID No. 115 GCATTTCTTCCATCTCCCCCTCuGCCT-SpC3
hACACA-Rev SEQ ID No. 116 TCCGATTCTTGCTCCACTGTTG
hACACA-Rev-rG SEQ ID No. 117 TCCGATTCTTGCTCCACTGTTGgCTGA-SpC3
03431 PCR reactions were done in 384 well format using a Roche Lightcycicr
480 platform. Reactions comprised lx BIO-RAD iQTM SYBR Green Supermix
(BIO-RAD, Hercules, CA), 200 nM of each primer (For + Rev), and 1.3 mU of
Pyrococcus abyssl RNasc 142 in 10 l_t1 volume. Template DNA was either 2 ng of
human HeLa cell cDNA or 2 ng of rat spinal cord cDNA. Thermal cycling
parameters
included an initial 5 minutes soak at 95 C and then 60 cycles were performed
of [95 C
for 10 seconds + 60 C for 90 seconds]. Under these conditions, the Cp value
observed
for human cDNA represents a true positive event. If any signal was detected
using rat
cDNA, it was recorded as a false positive event. For these 3 genes, the human
and rat
sequences are divergent at the primer binding sites. Therefore detection of a
PCR
product in rat cDNA using human gene specific primers is an undesired, false
positive
result that originates from mispriming. Results are shown in Table 26 below.
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CA 02949315 2016-11-23
Table 26: False detection of products in rat cDNA using human gene specific
primers
in a 60 cycle qRT-PCR reaction
Observed Cp Observed Cp
Primers (For/Rev) ACp
Human cDNA Rat cDNA
ETS2 23.6 56.4 32.8
ETS2-blocked 24.9 ND > assay
HRAS 25.2 35.5 10.3
HRAS-blocked 26.1 ND > assay
ACACA 26.2 52.3 26.1
ACACA-blocked 26.3 ND > assay
ND = not detected
103441 Using unmodified primers, detection of the human targets in human
cDNA
was successful and Cp's of 23-26 were observed. For all 3 PCR assays, the
human
gene-specific primers also detected products in rat cDNA when cycling was
continued,
and Cp's of 35-56 were observed. These represent undesired false positive
signals
which limit the ability of the PCR assays to detect low levels of true
positive signal.
[0345] Using modified primers, detection of the desired product in human
cDNA
was successful and Cp's were all within 1 of the values obtained for
unmodified
primers. However, no false positive signals were seen using rat cDNA with the
modified primers, even at 60 cycles. Use of the RNase H2 blocked-cleavable
primers
resulted in improved specificity, permitting use of longer, more sensitive PCR
reactions
(in this case up to 60 cycles) without detection of false priming events. This
allows for
a much greater ability to detect variant alleles in the presence of a larger
excess of the
wild type sequence.
EXAMPLE 12 ¨ Mismatch discrimination for a rC substrate under steady state
conditions
103461 Example 11 demonstrated the ability of the methods of the invention
to
improve specificity of a qPCR reaction in the face of background mispriming
events.
The present example demonstrates the specificity of the RNase H2 cleavage
reaction
with respect to single-base differences (SNPs). The ability of the Pyrococcus
abyssi
102
CA 02949315 2016-11-23
RNase H2 enzyme to distinguish base mismatches in a duplex substrate
containing a
single rC base was tested under steady state conditions. The following
substrates were
32P-end labeled and incubated in "Mg Cleavage Buffer" as described in Example
4
above. Reactions comprised 100 nM substrate with 100 uU of enzyme in 20
volume and were incubated at 70 C for 20 minutes. Reaction products were
separated
using denaturing 7M urea, 15% polyacrylamide gel electrophoresis (PAGE) and
visualized using a Packard Cyclone'1m Storage Phosphor System
(phosphorimager).
The relative intensity of each band was quantified and results plotted as a
fraction of
total substrate cleaved.
103471 Ten duplexes were studied, including the perfect match (rC:G, SEQ ID
No.
13 as well as each possible base mismatch at the rC base (3 duplexes, SEQ ID
Nos.
118-120), at position +1 relative to the rC (3 duplexes, SEQ ID Nos. 121-123),
and at
position -1 relative to the rC (3 duplexes, SEQ ID Nos. 124-126). Results were
normalized for perfect match = 100% and are shown in Table 27 below.
Table 27: Cleavage of rC substrates with and without mismatches under steady
state
conditions
Duplex Identity Substrate Sequence Cleavage
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 13100%
3' GAGCACTCCACTACGTCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 11846%
3' GAGCACTCCACTACATCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 119 3' GAGCACTCCACTACTTCCTCTACCCTCCGC 5' 35%
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 12023%
3' GAGCACTCCACTACCTCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 12119%
3' GAGCACTCCACTAAGTCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 12265%
3' GAGCACTCCACTATGTCCTCTACCCTCCGC 5'
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CA 02949315 2016-11-23
Table 27: Cleavage of rC substrates with and without mismatches under steady
state
conditions
Duplex Identity Substrate Sequence Cleavage
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 123 22%
3' GAGCACTCCACTAGGTCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 12461%
3' GAGCACTCCACTACGACCTCTACCCTCCGC 5'
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 125 91%
3' GAGCACTCCACTACGCCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGATGcAGGAGATGGGAGGCG 3'
SEQ ID No. 126 46%
3' GAGCACTCCACTACGGCCTCTACCCTCCGC 5'
DNA bases are shown as uppercase. RNA bases are shown as lowercase. Mismatches
are shown in bold font and are underlined.
103481 Pyrococcus RNasc H2 was able to discriminate between single base
mismatches under these conditions. The precise degree of discrimination varied
with
which bases were paired in the mismatch. Interestingly, mismatches at position
-1 (one
base 5' to the rC base) showed relatively good mismatch discrimination while
mismatches at position +1 (one base 3' to the rC base) were in general less
effective.
Although the selectivity appears relatively modest, it becomes greatly
amplified with
repeated cycles of PCR.
EXAMPLE 13 ¨ Mismatch discrimination for rN substrates during thermal
cycling
103491 The ability of the Pyrococcus abyssi RNase H2 enzyme to distinguish
base
mismatches for a rC substrate under steady state conditions was described in
Example
12. The ability of this enzyme to distinguish base mismatches for all rN
containing
substrates under conditions of thermal cycling was examined in the present
example.
In these conditions, the cleavable substrate is only available for processing
by the
enzyme for a short period of time before temperature elevation disrupts the
duplex.
Mismatch discrimination was assessed in the setting of a fluorescent
quantitative
real-time PCR assay. We found that base mismatch discrimination was greatly
104
CA 02949315 2016-11-23
improved under these kinetically limited conditions than were observed under
steady-state conditions.
[0350] The following nucleic acids were employed in this example.
Oligonucleotides were synthesized to provide coverage for all nearest neighbor
pairs
and mismatches.
Unmodified For primer:
103511 SEQ ID No. 86
5' AGCTCTGCCCAAAGATTACCCTG 3'
103521 Blocked rN substrate rev primers (C3 spacer blocking group at the 3'-
end)
are shown below. DNA bases are uppercase and RNA bases are lower case. Regions
of
variation are indicated by bold and underlined. At total of 28 blocked primers
containing a single RNA residue were synthesized.
rA series:
[0353] SEQ ID No. 127
5' CTGAGCTTCATGCCTTTACTGTaCCCC-SpC3 3'
[0354] SEQ ID No. 128
5' CTGAGCTTCATGCCTTTACTGAaCCCC-SpC3 3'
103551 SEQ ID No. 129
5' CTGAGCTTCATGCCTTTACTGCaCCCC-SpC3 3'
[0356] SEQ ID No. 130
5' CTGAGCTTCATGCCTTTACTGGaCCCC-SpC3 3'
[0357] SEQ ID No. 131
5' CTGAGCTTCATGCCTTTACTGTaTCCC-SpC3 3'
103581 SEQ ID No. 132
5' CTGAGCTTCATGCCTTTACTGTaGCCC-SpC3 3'
[0359] SEQ ID No. 133
5' CTGAGCTTCATGCCTTTACTGTaACCC-SpC3 3'
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rU series:
[0360] SEQ ID No. 134
5' CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 3'
[0361] SEQ ID No. 135
5' CTGAGCTTCATGCCTTTACTGAuCCCC-SpC3 3'
[0362] SEQ ID No. 136
5' CTGAGCTTCATGCCTTTACTGCuCCCC-SpC3 3'
[0363] SEQ ID No. 137
5' CTGAGCTTCATGCCTTTACTGGuCCCC-SpC3 3'
[0364] SEQ ID No. 138
5' CTGAGCTTCATGCCTTTACTGTuTCCC-SpC3 3'
[0365] SEQ ID No. 139
5' CTGAGCTTCATGCCTTTACTGTuGCCC-SpC3 3'
[0366] SEQ ID No. 140
5' CTGAGCTTCATGCCTTTACTGTuACCC-SpC3 3'
rC series:
[0367] SEQ ID No. 141
5' CTGAGCTTCATGCCTTTACTGTcCCCC-SpC3 3'
[0368] SEQ ID No. 142
5' cTGAGcTTcATGCcTTTACTGAcCCCC-SpC3 3'
[0369] SEQ ID No. 143
5' CTGAGCTTCATGCCTTTACTGCcCCCC-SpC3 3'
[0370] SEQ ID No. 144
5' CTGAGCTTCATGCCTTTACTGGcCCCC-SpC3 3'
103711 SEQ ID No. 145
5' CTGAGCTTCATGCCTTTACTGTcTCCC-SpC3 3'
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[0372] SEQ ID No. 146
5' CTGAGCTTCATGCCTTTACTGTcGCCC-SpC3 3'
[0373] SEQ ID No. 147
5' CTGAGCTTCATGCCTTTACTGTcACCC-SpC3 3'
rG series:
[0374] SEQ ID No. 148
5' CTGAGCTTCATGCCTTTACTGTgCCCC-SpC3 3'
[0375] SEQ ID No. 149
5' CIGAGCTTCATGCCTTTACTGAgCCCC-SpC3 3'
[0376] SEQ ID No. 150
5' CTGAGCTTCATGCCTTTACTGCgCCCC-SpC3 3'
[0377] SEQ ID No. 151
5' CTGAGCTTCATGCCTTTACTGGgCCCC-SpC3 3'
[0378] SEQ ID No. 152
5' CTGAGCTTCATGCCTTTACTGTgTCCC-SpC3 3'
[0379] SEQ ID No. 153
5' CTGAGCTTCATGCCTTTACTGTgGCCC-SpC3 3'
[0380] SEQ ID No. 154
5' CTGAGCTTCATGCCTTTACTGTgACCC-SpC3 3'
[0381] The unblocked control Rev primer (mimicing reaction product of
blocked
primers after cleavage by RN asc H2) employed was:
[0382] SEQ ID No. 87
5' CTGAGCTTCATGCCTTTACTG 3'
[0383] The following perfect-matched and mismatched synthetic templates
were
employed. The locations of varying bases are indicated in bold font with
underline.
Unique templates were made for each possible base variation at the
ribonucleotide or
one base 5' or one base 3' of the ribonucleotide. In total, 28 templates were
synthesized
and tested.
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CA 02949315 2016-11-23
rA templates:
[0384] SEQ ID No. 155
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTACAGTAAAGGCATGAAGCTCAG-3'
[0385] SEQ ID No. 156
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTCCAGTAAAGGCATGAAGCTCAG-3'
[0386] SEQ ID No. 157
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTTCAGTAAAGGCATGAAGCTCAG-3'
[0387] SEQ ID No. 158
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGTGCAGTAAAGGCATGAAGCTCAG-3'
[0388] SEQ ID No. 159
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCTACAGTAAAGGCATGAAGCTCAG-3'
[0389] SEQ ID No. 160
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGA7GTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGATACAGTAAAGGCATGAAGCTCAG-3'
[0390] SEQ ID No. 161
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCCGGTTACAGTAAAGGCATGAAGCTCAG-3'
rU templates:
[0391] SEQ ID No. 162
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
[0392] SEQ ID No. 163
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGATCAGTAAAGGCATGAAGCTCAG-3'
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CA 02949315 2016-11-23
[0393] SEQ ID No. 164
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGACCAGTAAAGGCATGAAGCTCAG-3'
[0394] SEQ ID No. 165
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGAGCAGTAAAGGCATGAAGCTCAG-3'
[0395] SEQ ID No. 166
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCAACAGTAAAGGCATGAAGCTCAG-3'
[0396] SEQ ID No. 167
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCC TCAGAAGTAGTGGCCAG
CTGTGTGTCGGGAAACAGTAAAGGCATGAAGC TCAG- 3'
[0397] SEQ ID No. 168
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGTAACAGTAAAGGCATGAAGCTCAG-3'
rG templates:
[0398] SEQ ID No. 169
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGCACAGTAAAGGCATGAAGCTCAG-3'
[0399] SEQ ID No. 170
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTCTCGGGGCTCAGTAAAGGCATGAACCTCAG-3'
[0400] SEQ ID No. 171
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGCCCAGTAAAGGCATGAAGCTCAG-3'
104011 SEQ ID No. 172
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGCGCAGTAAAGGCATGAAGCTCAG-3'
109
CA 02949315 2016-11-23
[0402] SEQ ID No. 173
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGACACAGTAAAGGCATGAAGCTCAG-3'
[0403] SEQ ID No. 174
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGTCACAGTAAAGGCATGAAGCTCAG-3'
[0404] SEQ ID No. 175
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCCACAGTAAAGGCATGAAGCTCAG-3'
rC templates
[0405] SEQ ID No. 176
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGACAGTAAAGGCATGAAGCTCAG-3'
[0406] SEQ ID No. 177
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGTCAGTAAAGGCATGAAGCTCAG-3'
[0407] SEQ ID No. 178
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGCCAGTAAAGGCATGAAGCTCAG-3'
[0408] SEQ ID No. 179
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGA7GTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGGGCAGTAAAGGCATGAAGCTCAG-3'
[0409] SEQ ID No. 180
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGAGACAGTAAAGGCATGAAGCTCAG-3'
[0410] SEQ ID No. 181
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGTGACAGTAAAGGCATGAAGC5CAG-3'
110
CA 02949315 2016-11-23
[0411] SEQ ID No. 182
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGCGACAGTAAAGGCATGAAGCTCAG-3'
[0412] Together, these nucleic acids comprise PCR assays set up as
indicated:
5'AGCTCTGCCCAAAGATTACCCTG 4
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG 3'
X-CCCCuTGTCATTTCCGTACTTCGAGTC 5'
TGTCATTTCCGTACTTCGAGTC 5'
[0413] The terminal C3 spacer group (indicated by "x") blocks the rU
containing
oligonucleotide to serve as a primer. When hybridized to the template, the
duplex
becomes a substrate for RNase H2 and cleavage occurs immediately 5'- to the rU
residue, resulting in a functional primer as shown
[0414] Quantitative real time PCR reactions were performed using unmodified
primer SEQ ID No. 86 and pairwise combinations of rN containing primers SEQ ID
Nos. 127-154 and templates SEQ ID Nos. 155-182. Reactions were done in 384
well
format using a Roche Lightcycler 480 platform. Reactions comprised lx BIO-RAD
iQTM SYBR Green Supermix (BIO-RAD, Hercules, CA), 200 nM of each primer (for
+ rev), and 1.3 mU of Pyrococcus abyssi RNase H2 in 10 IA volume. Thermal
cycling
parameters included an initial 5 minutes soak at 95 C and then 45 cycles were
performed of [95 C for 10 seconds + 60 C for 20 seconds + 72 C for 30
seconds].
Under these conditions, the Cp value was identical for control reactions done
using For
+ Rev (unmodified) primers and control coupled RNase H2 cleavage-PCR reactions
done using the perfect match For (unmodified) + rN Rev (blocked) primers. Thus
the
reaction conditions employed had sufficient incubation time and RNase H2
concentration to cleave the perfect match species within the kinetic
constraints of the
real time thermal cycling and any deviations from this point will represent a
change in
reaction efficiency imparted by base mismatches present between the blocked
primer
and the various templates.
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CA 02949315 2016-11-23
[0415] Pairwise combinations of primers and templates were run as described
above and results are summarized below showing ACp, which is the difference of
cycle
threshold observed between control and mismatch reactions. Since each Cp
represents
a cycle in PCR (which is an exponential reaction under these conditions), a
ACp of 10
represents a real differential of 210, or a 1024 fold change in sensitivity. A
ACp of 4 to
cycles is generally sufficient to discriminate between SNPs in allele specific
PCR
assays.
[0416] Results for tests done varying bases at the central position over
the rN base
are shown below in Table 28:
5' CTGAGCTTCATGCCTTTACTGTaCCCC-SpC3 3' blocked primers
3' GACTCGAAGTACGGAAATGACATGGGG_ 5' templates
A
Table 28. ACp for all possible base mismatches at the
rN position
Template
A
rA 14.9 9.4 13.6 0
rC 7.4 9.2 0 6.6
rG 13.9 0 12.7 14.5
rU 0 12.2 10.9 5.3
[0417] Very large differences in reactive efficiency are seen in RNase H2
cleavage
of a rN substrate under thermal cycling conditions, ranging from a difference
of around
40-fold (ACp 5.3) to over a 30,000 fold difference (ACp 14.9). None of the
assays
showed a ACp less than 5 cycles. Thus the RNase H2 rN cleavage reaction shows
far
greater specificity in the setting of a kinetic assay (qPCR) than under steady
state
conditions and much greater selectivity than allele specific PCR with standard
DNA
112
CA 02949315 2016-11-23
primers. Added specificity may be conferred by the design of the primers as
described
in the detailed description of the invention and demonstrated in the examples
below.
104181 Results for tests done varying bases at the -1 position relative to
the rN base
are shown below in Table 29:
A
5' CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 3' blocked primers
3' GACTCGAAGTACGGAAATGACAAGGGG . 5' templates
Table 29: ACp for all possible base mismatches at
position -1 relative to a rU base
Template
A
A(rU) 16.1 8.7 12.6 0
C(rU) 7.6 3.9 0 12.0
G(rU) 13.8 0 12.4 5.9
T(rU) 0 5.2 2.4 6.2
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[0419] Results for tests done varying bases at the +1 position relative to
the rN base
are shown below in Table 30:
A
5' CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 3' blocked primers
3' GACTCGAAGTACGGAAATGACAAGGGG. 5' templates
A
Table 30. ACp for all possible base mismatches at
position +1 relative to a rU base
Template
A
(rU)A 11.4 2.5 12.2 0
(rU)C 6.4 10.4 0 9.0
(rU)G 13.8 0 4.5 3.0
(rU)T 0 11.1 11.9 2.9
[0420] Pairwise combinations were similarly tested for all sequence
variants listed
above for the -1 and +1 positions relative to the rN base, including the rA,
rC, and rG
probes. Results arc shown in Tables 31-36 below.
Table 31: ACp for all possible base mismatches at position -1 relative to a rA
base
Template
A C G T
A(rA) 14.2 8.6 11.8 0
C(rA) 6.9 12.6 0 6.8
G(rA) 12.8 0 12.6 8.9
T(rA) 0 5.1 1.4 8.6
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Table 32: ACp for all possible base mismatches at position +1 relative to a rA
base
Template
A C G T
(rA)A 3.1 . 1.0 6.12 , 0
(rA)C 9.3 , 10.2 0 8.3
(rA)G 13.2 0 2.5 5.9
(rA)T 0 5.0 7.1 4.0
Table 33: ACp for all possible base mismatches at position -1 relative to a rC
base
-- -
Template
A C G T
A(rC) 13.0 8.2 10.5 0
C(rC) 5.0 3.3 0 3.5
G(rC) 8.3 0 7.0 0.8
T(rC) 0 5.4 2.1 4.6
Table 34: ACp for all possible base mismatches at position +1 relative to a rC
base
Template
A C G T
(rC)A 5.6 1.8 10.2 0
(rC)C 8.8 9.6 0 8.6
(rC)G 9.8 0 3.2 0.3
(rC)T 0 2.1 0.2 0.0
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Table 35: ACp for all possible base mismatches at position -1 relative to a rG
base
Template
A C
A(rG) 12.4 4.8 10.4 0
C(rG) 4.5 11.1 0 2.5
G(rG) 10.3 0 10.1 3.8
T(rG) 0 3.5 2.2 5.3
Table 36: ACp for all possible base mismatches at position +1 relative to a rG
base
Template
A
(rG)A 6.2 3.0 11.4 0
(rG)C 9.5 7.3 0 4.7
(rG)G 13.1 0 6.0 3.2
(rG)T 0 4.5 11.5 0.3
[0421] The relative change in reaction efficiency of cleavage of a rN
substrate by
Pyrococcus abyssi RNasc H2 in the setting of a single base mismatch varies
with the
identity of the paired bases, the relative position of the mismatch to the
cleavage site,
and the neighboring bases. The mismatch charts defined in this example can be
used to
design optimal mismatch detection assays which maximize the expected
differential
(ACp)between mismatch and matched loci, and can be built into an algorithm to
automate optimization of new assay designs.
Example 14 ¨ Mismatch discrimination for fUfU substrate under steady state
conditions
[0422] The ability of the Pyrococcus abyssi RNase H2 enzyme to distinguish
base
mismatches in a duplex substrate containing a fUfU dinucleotide pair was
tested under
steady state conditions. The following substrates were12P-end labeled and
incubated in
"Mn Cleavage Buffer" as described in Examples 5 and 6 above. Reactions
comprised
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CA 02949315 2016-11-23
100 nM substrate with 1 U of enzyme in 20 viL volume and were incubated at 70
C for
20 minutes. Reaction products were separated using denaturing 7M urea, 15%
polyacrylamide gel electrophoresis (PAGE) and visualized using a Packard
CycloneTM
Storage Phosphor System (phosphorimager). The relative intensity of each band
was
quantified and results plotted as a fraction of total substrate cleaved.
104231 Fourteen duplexes shown in Table 37, were studied, including the
perfect
match (SEQ ID No. 60), mismatches within the 2'-fluoro dinucleotide pair (SEQ
ID
Nos. 183-189), and mismatches adjacent to the 2'-fluoro dinucleotide pair (SEQ
ID
Nos. 190-195). Results were normalized for a perfect match = 100%.
Table 37: Cleavage of fUfU substrates with and without mismatches under steady
state
conditions
Duplex Identity Substrate Sequence Cleavage
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 60100%
3' GAGCACTCCACTA A A TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 1835%
3' GAGCACTCCACTA A G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 18414%
3' GAGCACTCCACTA A C TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 185 1%
3' GAGCACTCCACTA A T TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 1862%
3' GAGCACTCCACTA C T TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 187 0%
3' GAGCACTCCACTA G G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 1880%
3' GAGCACTCCACTA C C TCCTCTACCCTCCGC 5 '
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 1890%
3' GAGCACTCCACTA T T TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfU)AGGAGATGGGAGGCG 3'
SEQ ID No. 1908%
3' GAGCACTCCACTC A A TCCTCTACCCTCCGC 5'
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CA 02949315 2016-11-23
Table 37: Cleavage of fUfU substrates with and without mismatches under steady
state
conditions
Duplex Identity Substrate Sequence Cleavage
CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3
SEQ ID No. 191 4%
3 GAGCACTCCACTG A A
TCCTCTACCCTCCGC 5 '
5' CTCGTGAGGTGAT ( fUfU) AGGAGATGGGAGGCG 3 '
SEQ ID No. 192 4%
3 GAGCACTCCACTT A A TCCTCTACCCTCCGC 5
5
SE IDN 193 ' CTCGTGAGGTGAT ( fUfU)
AGGAGATGGGAGGCG 3' 2%
Q o.
3 GAGCACTCCACTA A A
GCCTCTACCCTCCGC 5 '
5
SE IDN 194 ' CTCGTGAGGTGAT ( fUfU)
AGGAGATGGGAGGCG 3 '
Q o. 8')/0
3 GAGCACTCCACTA A A
CCCTCTACCCTCCGC 5 '
5
SE ID 195 ' CTCGTGAGGTGAT ( fUfU)
AGGAGATGGGAGGCG 3'
Q No.
3 GAGCACTCCACTA A A ACCTCTACCCTCCGC 5( 2%
DNA bases are shown as uppercase. 2'-F bases are shown as fU.
Mismatches are shown in bold font and are underlined.
104241 Pyrococcus RNase 112 was able to discriminate very efficiently
between
single base mismatches under these conditions. The precise degree of
discrimination
varied with which bases were paired in the mismatch. Interestingly, mismatches
at both
positions -1 and +1 (relative to the fUfU domain) were effective. Specificity
for
cleavage using the fUfU substrate was significantly higher under steady state
assay
conditions than was the rC substrate (Example 12 above).
[0425] The study above
employed the fUfU dinucleotide pair, which was
previously shown in Example 6 to be the least efficient di-fluoro substrate
for cleavage
of the 16 possible dinucleotide pairs. This may impact the mismatch results.
Similar
experiments were conducted using the same complement strands, substituting a
fUfC
di-fluoro substrate strand. RNase H2 was reduced to 20 mU due to the increased
activity of cleavage seen for fUfC compared to fUfU substrates. Results are
shown in
Table 38 below.
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CA 02949315 2016-11-23
Table 38. Cleavage of flifC substrates with and without mismatches under
steady state
conditions
Duplex Identity Substrate Sequence Cleavage
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 58 100%
3' GAGCACTCCACTA A G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 1960%
3' GAGCACTCCACTA T G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 1977%
3' GAGCACTCCACTA C G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 1980%
3' GAGCACTCCACTA G G TCCTCTACCCTCCGC 5 '
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 199 1%
3' GAGCACTCCACTA A T TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2000%
3' GAGCACTCCACTA A C TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2012%
3' GAGCACTCCACTA A A TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2020%
3' GAGCACTCCACTA T C TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2030%
3' GAGCACTCCACTT A G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2044%
3' GAGCACTCCACTC A G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2050%
3' GAGCACTCCACTG A G TCCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2062%
3' GAGCACTCCACTA A G ACCTCTACCCTCCGC 5'
5' CTCGTGAGGTGAT(fUfC)AGGAGATGGGAGGCG 3'
SEQ ID No. 2074%
3' GAGCACTCCACTA A G CCCTCTACCCTCCGC 5'
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Table 38. Cleavage of fUfC substrates with and without mismatches under steady
state
conditions
Duplex Identity Substrate Sequence Cleavage
CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG 3'
SEQ ID No. 208 2%
3' GAGCACTCCACTA A G GCCTCTACCCTCCGC 5'
DNA bases are shown as uppercase. 2'-F bases are shown as fU.
Mismatches are shown in bold font and are underlined.
104261 Again, Pyrococcus abyssi RNase H2 was able to discriminate very
efficiently between single base mismatches. The precise degree of
discrimination
varied with which bases were paired in the mismatch. As before, mismatches at
both
positions -1 and +1 (relative to the fUfC domain) were effective. Specificity
for
cleavage using the fUfC substrate was significantly higher under steady state
assay
conditions than was the rC substrate (Example 12 above) and also showed
slightly
greater specificity than the fUfU substrate. Under kinetic assay conditions
during
thermal cycling, mismatch assays using di-fluoro substrates may show even
greater
selectivity.
Example 15 ¨ Selective placement of phosphorothioate internucleotide
modifications in the substrate
104271 The effect of incorporation of a phosphorothioate intemucleoside
linkage
was tested for several different substrates. Phosphorothioate (PS) bonds are
typically
considered relatively nuclease resistant and are commonly used to increase the
stability
of oligonucleotides in nuclease containing solutions, such as serum. PS bonds
form
two stereoisomers, Rp and Sp, which usually show different levels of
stabilization for
different nucleases.
104281 The di-fluoro substrate was examined with a PS bond between the two
modified bases. A mixture of both diastereomers was employed for the present
study.
Unmodified fUfC substrate:
104291 SEQ ID No. 58
5' -CTCGTGAGGTGAT ( fUfC) AGGAGATGGGAGGCG- 3 '
3' -GAGCACTCCACTA A G TCCTCTACCCTCCGC-5
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PS modified fU*fC substrate ("*" = PS bond):
[0430] SEQ ID No. 209
5'-CTCGTGAGGTGAT(fU*fC)AGGAGATGGGAGGCG-3'
3'-GAGCACTCCACTA A G TCCTCTACCCTCCGC-5'
(note ¨ gaps in sequence are for alignment purposes)
104311 The above substrates were incubated for 1 hour at 70 C in "Mn
Cleavage
Buffer" using 160 pmoles of substrate in 120 ul volume (1.3 p,M) and 4 units
of the
recombinant Pyrococcus RNasc 142 enzyme. Reactions were stopped with the
addition
of gel loading buffer (formamide/EDTA) and separated on a denaturing 7M urea,
15%
polyacrylamide gel. Gels were stained using GelStarTM (Lonza, Rockland, ME)
and
visualized with UV excitation. The unmodified substrate was 100% cleaved under
these conditions; however the PS-modified substrate was essentially uncleaved.
The
phosphorothioatc modification can effectively block cleavage of a di-fluoro
substrate.
[0432] A substrate containing a single rC residue was studied next, testing
placement of the PS modification on either side of the RNA base (5'- or 3'-
side as
indicated). A mixture of both diastereomers were employed for the present
study.
Unmodified rC substrate:
[0433] SEQ ID No. 13
5f-CTCGTGAGGTGATTcAGGAGATGGGAGGCG-3'
3'-GAGCACTCCACTAAGTCCTCTACCCTCCGC-5'
PS modified 5' *rC substrate:
[0434] SEQ ID No. 210
5'-CTCGTGAGGTGATT*cAGGAGATGGGAGGCG-3'
3f-GAGCACTCCACTAA GTCCTCTACCCTCCGC-5'
PS modified 3' rC* substrate:
[0435] SEQ ID No. 211
5'-CTCGTGAGGTGATTc*AGGAGATGGGAGGCG-3'
3'-GAGCACTCCACTAAG TCCTCTACCCTCCGC-5'
[0436] The above substrates were incubated for 1 hour at 70 C in "Mg
Cleavage
Buffer" using 160 pmoles of substrate in 120 ill volume (1.3 iiiM) and 4 units
of the
recombinant Pyrococcus RNase H2 enzyme. Reactions were stopped with the
addition
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of gel loading buffer (formamide/EDTA) and separated on a denaturing 7M urea,
15%
polyacrylamide gel. Gels were stained using GelStarrm (Lonza, Rockland, ME)
and
visualized with UV excitation. The unmodified substrate was 100% cleaved under
these conditions. Both the 5'-*rC
and 3'-rC* PS-modified substrates were
approximately 50% cleaved under these conditions. These results arc most
consistent
with one stereoisomer, Rp or Sp, being more resistant to cleavage than the
other isomer.
104371 The 3'-rC*
substrate was studied in greater detail. Since RNase H2 cleaves
this substrate on the 5 '-side of the ribonucleotide while other RNases (such
as RNase A,
RNase 1, etc.) cleave this substrate on the 3'-side of the ribonucleotide, it
may be
possible to use the PS modification as a way of protecting the substrate from
unwanted
degradation by other nucleases while leaving it available as an RNase H2
substrate. It
is well known that cleavage of RNA substrates by RNase A and other single-
stranded
ribonucleases is inhibited to a greater extent by the Sp phosphorothioate
isomer than the
Rp isomer. The relative effects of the Sp vs. Rp isomer on RNase H2 cleavage
have not
been known. Therefore the two stercoisomers were purified and the relative
contributions of the Sp and Rp isomers on 3'-rC* substrate stability were
studied.
104381 It is well
known that phosphorothioate isomers can be separated by HPLC
techniques and that this separation is readily done if only a single PS bond
exists in an
oligonucleotide. HPLC was therefore employed to purify the two PS isomers of
the
3'-rC* substrate, SEQ ID No. 211. A mass of 7 nmoles of the single-stranded 3'-
rC*
containing oligonucleotide was employed. Characterization showed that the test
material had a molecular weight of 9464 Daltons (calculated 9465) by ESI-MS
with a
molar purity of 95% by capillary electrophoresis. This material was injected
into a 4.6
mm x 50 mm Xbridge im C 18 column (Waters) with 2.5 micron particle size.
Starting
mobile phase (Buffer A) was 100 mM TEAA pH 7.0 with 5% acetonitrile and which
was mixed with pure acetonitrile (Buffer B) at 35 C. The HPLC method employed
clearly resolved two= peaks in the sample which were collected and re-run to
demonstrate purity. HPLC traces of the mixed isomer sample and purified
specimens
are shown in Figure 23. Both the "A" and "B" peaks had an identical mass of
9464
Daltons by ESI-MS. From the original sample, 1.3 nmoles of peak "A" and 3.6
nmoles
of peak "B" were recovered.
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104391 It was not possible based upon mass or HPLC data to identify which
peak
was the Rp and which peak was the Sp isomer. Relative resistance to
degradation by
RNase A was employed to assign isomer identity to the purification fractions.
The Sp
isomer is known to confer relatively greater resistance to RNase A degradation
than the
Rp isomer. Purified products were studied in the single-stranded form. The
substrate
was radiolabeled with 32P using 6000 Ci/mmol y-32P-ATP and the enzyme T4
Polynucleotide Kinase (Optikinase, US Biochemical). Trace label was added to
reaction mixtures (1:50). Reactions were performed using 100 nM substrate in
20 1.11
volume with 1 pg (72 attomoles) of RNase A in Mg Cleavage Buffer. Reactions
were
incubated at 70 C for 20 minutes. Reaction products were separated using
denaturing
7M urea, 15% polyacrylamide gel electrophoresis (PAGE) and visualized using a
Packard CycloneTM Storage Phosphor System (phosphorimager). The relative
intensity
of each band was quantified and results plotted as a fraction of total
substrate cleaved.
Peak "A" was more completely degraded by RNase A than peak "B"; peak "A" was
therefore assigned identity as the Rp isomer and peak "B" was assigned as the
Sp
isomer.
104401 The relative susceptibility of each stereoisomer to RNase H2
cleavage was
studied. The RNA-containing strand of the substrate was radiolabeled with 32P
using
6000 Ci/mmol y-32P-ATP and the enzyme T4 Polynucleotide Kinase (Optikinase, US
Biochemical). Trace label was added to reaction mixtures (1:50). Reactions
were
performed using 100 nM substrate in 20 ,u1 volume with 100 U of recombinant
Pyrococcus abyssi RNase H2 in Mg Cleavage Buffer. Substrates were employed in
both single-stranded and duplex form. Reactions were incubated at 70 C for 20
minutes. Reaction products were separated using denaturing 7M urea, 15%
polyacrylamide gel electrophoresis (PAGE) and visualized using a Packard
Cyclone'rm
Storage Phosphor System (phosphorimager). The relative intensity of each band
was
quantified and results plotted as a fraction of total substrate cleaved. As
expected,
single-stranded substrates were not cleaved by the RNase H2 enzyme. The
control
unmodified rC duplex (SEQ ID No. 13) was 100% cleaved under the conditions
employed. The Sp isomer 3'-rC* duplex substrate (peak "B") was cleaved ¨30%
whereas the Rp isomer (peak "A") was cleaved < 10% under these conditions.
Therefore the relative susceptibility to cleavage of racemically pure
phosphorothioate
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CA 02949315 2016-11-23
modified substrates at this position (3'-to the ribonucleotide) is exactly
opposite for
RNase H2 vs. RNase A. The Sp isomer is more readily cleaved by RNase H2 while
the
Rp isomer is more readily cleaved by RNase A. Therefore single ribonucleotide
containing substrates having a racemically pure Sp isomer phosphorothioate
modification on the 3'-side of the ribonucleotide could be employed to protect
this
bond from unwanted degradation by single-stranded nucleases (such as RNase A)
while still being a functional substrate for cleavage by RNase H2. The
relationship
between enzyme cleavage and phosphorothioate stereoisomer is summarized in
Figure
24.
EXAMPLE 16 ¨ Utility of rN containing dual-labeled probes in qPCR assays
[0441] The following example illustrates a real time PCR assay utilizing a
rU-containing dual labeled probe. Previously, we demonstrated in Example 9 the
feasibility for use of rN blocked primers in qPCR using a SYBR3') Green
detection
format. Cleavage of blocked oligonucleotides using the method of the present
invention can also be applied to the dual-labeled probe assay format. Use of
RNase HI
to cleave a dual-labeled probe containing a 4 RNA base cleavage domain in an
isothermal cycling probe assay format has been described by Harvey, J.J., et
al.
(Analytical Biochemistry, 333:246-255, 2004). Another dual-labeled probe assay
using RNase H has been described, wherein a molecular beacon containing a
single
ribonucleotide residue was employed to detect polymorphisms in an end-point
PCR
format using RNase H2 (Hou, J., et al., Oligonucleotides, 17:433-443, 2007).
In the
present example we will demonstrate use of single ribonucleotide containing
dual-labeled probes in a qPCR assay format that relies upon RNase H2 cleavage
of the
probe.
[0442] The following oligonucleotides shown in Table 39, were used as
probes and
primers in a qPCR assay with a dual-labeled fluorescence-quenched probe. The
target
was a synthetic oligonucleotide template.
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Table 39:
Name Sequence SEQ ID No.
Syn-For 5'-AGCTCTGCCCAAAGATTACCCTG-3' SEQ ID No. 86
Syn-Rev 5'-CTGAGCTTCATGCCTTTACTGT-3' SEQ ID No. 87
Syn-Probe 5'-FAM-TTCTGAGGCCAACTCCACTGCCACTTA-IBFQ-3' SEQ ID No. 212
Syn-Probe-rU 5 - FAM- T T C TGAGGCCAACuCCAC TGCCAC T TA - BFQ - 3 SEQ ID No.
213
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein and IBFQ is a dark quencher (Integrated DNA
Technologies).
[0443] Synthetic template (primer and probe binding sites are underlined).
[0444] SEQ ID No. 93
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0445] Quantitative real time PCR reactions were performed using unmodified
primers SEQ ID Nos. 86 and 87 and probes Seq ID Nos. 212 and 213. Reactions
were
done in 384 well format using a Roche Lightcycler 480 platform. Reactions
comprised 200 nM of each primer (for + rev) and 200 nM probe, 2 x 106 copies
of the
synthetic template, and 5 mU of Pyrococcus abyysi RNase H2 in 10 pi volume.
Thermal cycling parameters included an initial 10 minutes incubation at 95 C
and then
45 cycles were performed of [95 C for 10 seconds + 60 C for 30 seconds + 72 C
for 1
seconds]. The buffer employed varied with the polymerase used.
[0446] If PCR is performed using a thermostable DNA polymerase having
5'-exonuclease activity the polymerase will degrade the probe. Under these
conditions,
a DNA probe should perform the same as a rN modified probe. This reaction
constitutes a positive control. If a DNA polymerase is employed which is
lacking
5'-exonuclease activity, then neither probe should be degraded. This reaction
constitutes a negative control. A PCR reaction using the exo-negative
polymerase with
RNase H2, however, should degrade the rN containing probe but not the DNA
probe,
demonstrating function of the invention. For the present study, the following
two
thermostable polymerases were used: Immolase (intact 5' nuclease activity,
Bioline)
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and Vent Exo- (5'-exonuclease negative mutant, New England Biolabs). Buffers
employed were the manufacturer's recommended buffers for the DNA polymerases
and were not optimized for RNase H2 activity. For Immolase, the buffer
comprised 16
mM (NH4)2SO4, 67 mM Tris pH 8.3, and 3 mM MgC12. For Vent Exo-, the buffer
comprised 10 mM (NH4)2SO4, 20 mM Tris pH 8.8, 10 mM KC1, and 3 mM MgSO4.
104471 qPCR reactions were run as described and results are shown below in
Table
40.
Table 40. Cp values of qPCR reactions comparing DNA or rU Dual-Labeled Probes
Probe Polymerase Minus RNase 112 Plus RNase H2
Syn-Probe Immolase Exo 21.1 21.0
Vent Exo- ND ND
Syn-Probe-rU Immolase Exo' 21.0 20.7
Vent Exo- ND 21.1
ND = not detectable
[0448] Using the exonuclease positive polymerase, both probes showed
similar
functional performance and gave similar Cp values, both with or without RNase
H2.
Using the exonuclease deficient mutant polymerase, however, the DNA probe did
not
produce any detectable fluorescent signal; the rU probe failed to produce
fluorescent
signal in the absence of RNase H2, but in the presence of RNase H2 was cleaved
and
resulted in signal at the expected Cp value. Similar results can be obtained
using
di-fluoro containing probes. If the RNase H2 cleavage domain is placed over a
mutation site such probes can be used to distinguish variant alleles.
104491 RNase H-cleavable probes can also be linked with the use of blocked
primers of the present invention to additively increase the specificity of
amplification
based assay systems.
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EXAMPLE 17 Utility of rN containing blocked primer to prevent primer-dimer
formation
104501 Formation of primer-dimers or other small target independent
amplicons
can be a significant problem in both endpoint and real-time PCR. These
products can
arise even when the primers appear to be well designed. Further, it is
sometimes
necessary to employ primers which have sub-optimal design because of sequence
constraints for selection of primers which hybridize to specific regions. For
example,
PCR assays for certain viruses can be subtype or serotype specific if primers
are chosen
in areas that are variable between strains. Conversely, PCR reactions can be
designed
to broadly amplify all viral strains if primers are placed in highly conserved
regions of
the viral genome. Thus the sequence space available to choose primers may be
very
limited and "poor" primers may have to be employed that have the potential to
form
primer dimers. Use of "hot start" PCR methods may eliminate some but not all
of these
problems.
104511 The following example derives from one such case cited in US Patent
06001611 where primer-dimers were found to be a significant problem during
development of a PCR-based nucleic acid detection assay for the Hepatitis C
virus
(HCV) using sites in conserved domains that permit detection of a wide range
of viral
serotypes. We demonstrate herein that use of cleavable blocked primers can
prevent
unwanted primer dimer formation, specifically in the absence of a "hot-start"
DNA
polymerase.
[0452] The following oligonucleotides, as shown in Table 41, were used as
primers
in a PCR assay. The target was a cloned synthetic amplicon isolated from a
plasmid.
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Table 41:
Name Sequence SEQ ID No.
ST280A-for 5'-GCAGAAAGCGTCTAGCCATGGCGTTA SEQ ID No. 214
ST778AA-rev 5'-GCAAGCACCCTATCAGGCAGTACCACAA SEQ ID No. 215
ST280A-for-B 5' -GCAGAAAGCGTCTAGCCATGGCGTTAgTATG-SpC 3 SEQ ID No. 216
ST778AA-rev-B 5' -GCAAGCACCCTATCAGGCAGTACCACAAgGCCT- SpC 3 SEQ ID No. 217
DNA bases are shown in uppercase. RNA bases are shown in lowercase. SpC3 is a
C3
spacer. The "B" designation indicates a blocked, cleavable primer.
Cloned synthetic target (primer binding sites are underlined).
[0453] SEQ ID No. 218
Hepatitis C virus subtype lb amplicon (242 bp):
gcagaaagegtetagccatggegttagtatgagtgtcgtgcagcctccaggacccoccotcccgggaga
gccatagtggtctgcggaaccggtgagtacaccggaattgccaggacgaccgggtcctttcttggacta
aacccgctcaatgcctggagatttgggcgtgcccccgcgagactgctagccgagtagtgttgggtcgcg
aaaggccttgtggtactgcctgatagggtgcttgc
[0454] PCR reactions were done in 384 well format using a Roche Lightcycler
480 platform. Reactions comprised lx New England Biolabs (Beverly, MA) DyNAmo
reaction mix with DyNAmo DNA polymerase, 200 nM of each primer (For + Rev),
with or without 1.3 mU of Pyrococcav abysm' RNase H2 in 10 ill volume.
Template
DNA was either 2000 copies of the linearized HCV plasmid amplicon or no target
control. Thermal cycling parameters included an initial 2 minutes soak at 95 C
and
then 50 cycles were performed of [95 C for 15 seconds + 60 C for 30 seconds].
Samples were separated on an 8% polyacrylamide non-denaturing gel and
visualized
using GelStar stain. Results are shown in Figure 25. The unblocked standard
primers
produced multiple products having sizes ranging from 55 bp to 90 bp in size
and no
desired full length product was seen. In the absence of RNase H2, use of the
blocked
primers did not result in any amplified product. With RNase H2, the blocked
primers
produced a single strong amplicon of the expected size and no undesired small
species
were seen.
[0455] The DyNAmo is a non hot-start DNA polymerase. Use of RNase H2
blocked primer of the present invention with a hot-start RNase H2 having
reduced
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activity at lower temperatures eliminated undesired primer-dimers from the
reaction
and resulted in formation of the desired amplicon whereas standard unblocked
primers
failed and produced only small, undesired species.
EXAMPLE 18 Use of detergent in RNase H2 assay buffers
104561 The presence of
detergent was found to be beneficial to cleavage by the
Pyrococcus abyssi RNase H2 enzyme. Different detergents were tested at
different
concentrations to optimize the reaction conditions.
[0457] Aliquots of each of
the recombinant RNase H2 enzymes were incubated
with the single-stranded and double-stranded oligonucicotide substrates
indicated
above in an 80 pl reaction volume in buffer 50 mM NaC1, 10 mM MgCl2, and 10 mM
Tris pH 8.0 for 20 minutes at 70 C. Reactions were stopped with the addition
of gel
loading buffer (formamide/EDTA) and separated on a denaturing 7M urea, 15%
polyacrylamidc gel. The RNA strand of the substrate SEQ ID No. 13 was
radiolabeled
with 32P. Reactions were performed using 100 nM substrate with 100 microunits
(tI)
of enzyme in Mg Cleavage Buffer with different detergents at varying
concentrations.
TM
Detergents tested included Triton-X100, Tween-20, Tween-80, CTAB, and N-
lauryol
sarcosyl. Results with Pyrococcus absii RNase H2 are shown in Figure 26.
Additional
experiments were done to more finely titrate CTAB detergent concentration.
Optimum
levels of detergent to obtain highest enzyme activity were (vol:vol): Triton-
X100
0.01%, Tween-20 0,01%, and CTAB 0.0013%. The detergents Tween-80 and
N-lauryol sarcosyl did not perform as well as the other detergents tested.
Thus both
non-ionic (Triton. Tween) and ionic (CTAB) detergents can be employed to
stabilize
thermophilic RNasc H2 enzymes of the present invention.
EXAMPLE 19 Use of fluorescence-quenched (F/Q) cleavable primers in qPCR
104581 In Example 9 above,
it was demonstrated that cleavable blocked primers
function in PCR and further can be employed in real-time quantitative PCR
(qPCR)
using SYBR green detection. In this example we
demonstrate use of
fluorescence-quenched cleavage primers where the primer itself generates
detectable
signal during the course of the PCR reaction.
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[0459] Figure 18 illustrates the scheme for performing PCR using blocked
cleavable primers. Figure 27 illustrates the scheme for performing PCR using
fluorescence-quenched cleavable primers. In this case one primer in the pair
is
detectably labeled with a fluorescent dye. A fluorescence quencher is
positioned at or
near the 3'-end of the primer and effectively prevents priming and DNA
synthesis when
the probe is intact. A single ribonucleotide base is positioned between the
dye and the
quencher. Cleavage at the ribonucleotide by RNase H2 separates the reporter
and
quencher, removing quenching, resulting in a detectable signal. Concomitantly,
cleavage activates the primer and PCR proceeds.
[0460] The following synthetic oligonucleotides shown in Table 42, were
employed to demonstrate this reaction using a synthetic template. As a control
the
5'-nuclease Taqmae assay was performed with unmodified primers and a standard
fluorescence-quenched probe. Three variants of the synthetic fluorescence-
quenched
cleavable primers were compared, having 4, 5, or 6 DNA bases 3' to the RNA
base. It
was previously established that 4 DNA bases 3' to the RNA base was optimal
using
oligonucleotide substrates having a C3 spacer or ddC end group. It was
possible that
the presence of a bulky hydrophobic quencher group at or near the 3'-end might
change
the optimal number of DNA residues needed in this domain.
Table 42:
Name Sequence SEQ ID No.
Syn-For 5r-AGCTCTGCCCAAAGATTACCGTG SEQ ID No. 86
Syn-Rev 5r-CTGAGCTTCATGCCTTTACTGT SEQ ID No. 87
Syn-Probe 5'-FAM-TTCTGAGGCCAACTCCACTGCCACTTA-IBFQ SEQ ID No. 219
Syn-For
5f-FAM-CTGAGCTTCATGCCTTTACTGTuCCCC-IBFQ SEQ ID No. 220
F/Q-4D
Syn-For
5'-FAM-CTGAGCTTCATGCCTTTACTGTuCCCCG-IBFQ SEQ ID No. 221
F/Q-5D
Syn-For
5r-FAM-CTGAGCTTCATGCCTTTACTGTuCCCCGA-IBFQ SEQ ID No. 222
F/Q-6D
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher.
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Synthetic template
[0461] SEQ ID No. 93
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0462] PCR reactions were performed in 10 1 volume using 200 nM primers,
200
M of each dNTP (800 M total), 1 unit of iTaq (BIO-RAD), 50 mM Tris pH 8.3, 50
mM KC1, and 3 mM MgC12. Reactions were run either with or without varying
amounts of P yrOCOCCUS abyssi RNasc H2 on a Roche Lightcycler 480 platform
with 2
x 106 copies of synthetic template/target oligonucleotidc (SEQ ID No. 93).
Reactions
were started with a soak at 95 C for 5 minutes followed by 45 cycles of 195 C
for 10
seconds, 60 C for 30 seconds, and 72 C for 1 second]. The For and Rev primers
(SEQ
ID Nos. 86 and 87) were used with the internally placed DLP (SEQ ED No. 219).
Alternatively, the For primer (SEQ ID No. 86) was used with the FQ primers
(individually) (SEQ ID Nos. 220-222).
[0463] Use of the F/Q cleavable primers resulted in detectable fluorescence
signal
in real time during PCR similar to that obtained using the traditional dual-
labeled probe
(DLP) (SEQ ID No. 219) in the 5'-nuclease assay format. Primer SEQ ID No. 220,
with 4 DNA residues 3' to the RNA base, showed delayed amplification relative
to the
unmodified primers. Primers SEQ ID No. 221 and 222, with 5 and 6 DNA bases 3'
to
the RNA base, were more efficient and performed equally well. It is therefore
preferable to use an oligonucleotide design with 5 DNA bases 3' to the RNA
base in
this assay format as opposed to the 3-4 DNA base design optimal when the 3'-
blocking
group is smaller. In previous Examples using a SYBR Green assay format, 1.3 mU
of
RNase I-12 resulted in priming efficiency identical to unmodified primers. In
the
present F/Q assay format, use of 1.3 mU of RNase H2 resulted in delayed
amplification
whereas use of 2.6 mU of RNase H2 resulted in identical results compared to
unmodified primers. Increasing the amount of RNase H2 for the F/Q assay format
is
therefore preferred. Both amplification and detection of signal was RNase H2
dependent.'
[0464] Examples of amplification plots for qPCR reactions run using the
5'-nuclease assay DLP (SEQ ID No. 219) and the F/Q cleavable 5D primer (SEQ ID
131
CA 02949315 2016-11-23
No. 221) are shown in Figure 28. It is evident that amplification efficiency
is similar
between both methods as the Cp values where fluorescence is first detected is
identical
(20.0). Interestingly, the ARf (the magnitude of fluorescence signal detected)
peaked at
slightly higher levels using the DLP than the FQ primer. One possible
explanation for
the difference in maximal fluorescence signal release is that the fluorescent
dye on the
FQ primer remained partially quenched at the end of the reaction. In the 5'-
nuclease
assay, the probe is degraded and the reporter dye is released into the
reaction mixture
attached to a single-stranded short nucleic acid fragment. In the FQ primer
assay
format the fluorescent reporter dye remains attached to the PCR amplicon and
is in
double-stranded format. DNA can quench fluorescein emission, so this
configuration
might lower the final signal.
10465] We therefore tested if changing dye/quencher configuration on the
primer
would alter the fluorescence signal, comparing F/Q vs. Q/F versions of the
same primer.
In the synthetic amplicon assay used above, the preferred 5-DNA probe has a
residue present at the G residues tend to quenche FAM, whereas other bases
have little effect on FAM fluorescence. The amplicon was therefore modified to
change this base. The sequences in Table 43, were synthesized and tested in a
fluorescent real-time PCR assay format.
Table 43:
Name Sequence SEQ ID No.
Syn-For 5'-AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 86
Syn-For(C)
5'-FAM-CTGAGCTTCATGCCTTTACTGTuCCCCC-IBFQ SEQ ID No. 223
F/Q-5D
Syn-For(C)
5'-IBFQ-CTGAGCTTCATGCCTTTACTGTuCCCCC-FAM SEQ ID No. 224
Q/F-5D
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher.
Synthetic template
104661 SEQ ID No. 225
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTGGGGGAACAGTAAAGGCATGAAGCTCAG
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[0467] PCR reactions were performed in 10 IA volume using 200 nM primers,
200
JIM of each dNTP (800 !.11V1 total), 1 unit of iTaq (BIO-RAD), 50 mM Tris pH
8.3, 50
mM KC1, and 3 mM MgC12. Reactions were run with 2.6 mU of Pyrococcus abyssi
RNase H2 on a Roche Lightcycler 480 platform with 2 x 106 copies of synthetic
template/target oligonucleotide (SEQ ID No. 225). Reactions were started with
a soak
at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60 C for
30
seconds, and 72 C for 1 second]. The For primer (SEQ ID No. 86) was used with
either
the FQ primer (SEQ ID No. 223) or the QF primer (SEQ ID No. 224).
[0468] Use of the F/Q and Q/F cleavable primers resulted in an identical
Cp,
indicating that both primers performed with equal efficiency in the reaction.
As
predicted, the Q/F primer showed increased ARf relative to the F/Q primer.
Both
versions of the primer work equally well in the assay.
EXAMPLE 20 Use of fluorescence-quenched (F/Q) cleavable primers in multiplex
qPCR
[0469] Multiplex assays are commonly employed today to streamline
experiments
and increase throughput. It is particularly common to combine a qPCR assay
specific
for an experimental gene of interest with a second qPCR assay specific for an
internal
reference control gene for normalization purposes. One weakness of SYBR Green
detection for qPCR is that multiplex reactions are not possible. The use of
dye-labeled
fluorescence-quenched probes or primers does permit such multiplex reactions
to be
run. Real time PCR cycling and detection equipment is available today that
permits
combination of 2, 3, or 4 different fluorophores into the same reaction tube.
This
example demonstrates the utility of fluorescence-quenched (F/Q) cleavable
primers in
multiplex qPCR.
[0470] The following oligonucleotide reagents shown in Table 44, were
synthesized to perform multiplex qPCR using either a dual-labeled probe with
the
5'-nuclease assay or an F/Q cleavable primer. One assay was specific for the
human
MYC gene (NM 002476) and the second assay was specific for the human SFRS9
gene (NM_003769), a splicing factor which is a commonly used internal
normalization
control gene.
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Table 44:
Name Sequence SEQ ID No.
MYC-For 5' -TCGGATTCTCTGCTCTCCT SEQ ID No. 226
MYC-Rev 5f-CCTCATCTTCTTGTTCCTCC SEQ ID No. 227
MYC-Probe 5f-FAM-CCACCACCAGCAGCGACTCTGA-IBFQ SEQ ID No. 228
MYC-For-FQ 5 -FAM-TCGGATTCTCTGCTCTCCTcGACGG- I BFQ SEQ ID No. 229
MYC-Rev-B 5 -CCTCATCTTCTTGTTCCTCCuCAGA- SpC3 SEQ ID No. 230
SFRS9-For 5' -TGTGCAGAAGGATGGAGT SEQ ID No. 231
SFRS9-Rev 5' -CTGGTGCTTCTCTCAGGATA SEQ ID No. 232
SFRS9-Probe 5 -MAX- TGGAATATGCCCTGCGTAAAC TGGA- I BFQ SEQ ID No. 233
SFRS9-For-FQ 5' -MAX- TGTGCAGAAGGATGGAGTgGGGAT- I BFQ SEQ ID No. 234
SFRS9-Rev-B 5' -CTGGTGCTTCTCTCAGGATAaACTC - SpC 3 SEQ ID No. 235
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher. MAX is a red
reporter
dye. SpC3 is a C3 spacer.
[0471] PCR reactions were performed in 10 ul volume using 200 nM primers
(and
probe where appropriate), 200 M of each dNTP (800 IVI total), 1 unit of iTaq
(BIO-RAD), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM MgC12. Reactions were run
with 10 mU of PyrOCOCCILS abyssi RNase H2 on a Roche Lightcycler 480 platform
with 2 ng of cDNA made from total HeLa cell RNA. Reactions were started with a
soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60 C
for 30
seconds, and 72 C for 1 second].
10472] The multiplex reactions for the 5 '-nuclease assays included the MYC
For
and Rev primers + MYC probe (SEQ ID Nos. 226-228) and the SFRS9 For and Rev
primers + SFRS9 probe (SEQ ID Nos. 231-233). The multiplex reactions for the
FQ-cleavable primer assays included the MYC-For-FQ and MYC-Rev-B blocked
primers (SEQ ID Nos. 229 and 230) and the SFRS9-For-FQ and SFRS9-Rev-B blocked
primers (SEQ ID Nos. 234 and 235). All assays were also run in singleplex
format for
comparison. The FAM primers and probes were detected in the fluorescein dye
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CA 02949315 2016-11-23
channel while the MAX primers and probes were detected in the HEX dye channel.
Both the multiplexed DLP 5'-nuclease assays and the multiplexed FQ-cleavable
primer
assays worked well and resulted in very similar data, which is summarized in
Table 45
below.
Table 45. Multiplex qPCR reactions for MYC and SFRS9
Cp Value Cp Value
Reaction
FAM Channel HEX Channel
MYC FAM DLP 25.7
SFRS9 MAX DLP 24.8
MYC FAM DLP +
24.6 23.9
SFRS9 MAX DLP
MYC FAM FQ-Primer 27.2
SFRS9 MAX FQ Primer 28.0
MYC FAM FQ-Primer +
27.9 26.1
SFRS9 MAX FQ Primer
10473] RNasc H concentration was titrated and higher levels of enzyme were
needed to maintain reaction efficiency in multiplex format. For example,
blocked
primers in singleplex SYBR Green detection format required 1.3 mU of enzyme.
Blocked FQ primers in singleplex format required 2.6 mU of enzyme. Blocked FQ
primers in multiplex format required 10 mU of enzyme. It is therefore
important to
titrate the amount of RNase H2 enzyme employed when cleavable primers are used
in
different assay formats.
10474] Another application where use of multiplex probes is common practice
is
allelic discrimination SNPs. The following assay was designed to distinguish a
SNP
pair for the SMAD7 gene at a site that is known to be relevant for development
of
colorectal carcinoma, rs4939827. FQ blocked primers were designed and
synthesized
at this site using the standard design features taught in the above examples
without any
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CA 02949315 2016-11-23
further optimization to discriminate between the "C" and "T" alleles in this
gene.
Sequences are shown below in Table 46.
Table 46:
Name Sequence SEQ ID No.
rs4939827 Rev 5' -C TCAC TC TAAACCCCAGCATT SEQ ID No. 236
rs4939827
C-FAM-FQ-For 5' - FAM-CAGCCTCATCCAAAAGAGGAAAcAGGA- I BFQ SEQ ID No.237
rs4939827
T-HEX-FQ-For ' - HEX -CAGCC TCATCCAAAAGAGGAAAuAGGA- I BFQ SEQ ID No.238
DNA bases are shown in uppercase. RNA bases are shown in lowercase. FAM is
6-carboxyfluorescein. IBFQ is Iowa Black FQ, a dark quencher. MAX is a red
reporter
dye.
[0475] The above primers target the following 85 bp region of the SMAD7
gene
(NM 005904). Primer binding sites are underlined and the SNP location is
highlighted
as bold italic.
rs4939827 (SMAD7) C allele (SEQ ID No. 239)
CAGCC TCATCCAAAAGAGGAAACAGGACGCCAGAGC TCCC TCAGAC TCC TCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
rs4939827 (SMAD7) T allele (SEQ ID No. 240)
CAGCC TCATCCAAAAGAGGAAA TAGGACCCCAGAGC TCCC TCAGAC TCC TCAGGAAACACAGACAAT GC
TGGGGTTTAGAGTGAG
[0476] PCR reactions were performed in 10 ul volume using 200 nM FQ-For and
unmodified Rev primers, 200 nM of each dNTP (800 uM total), 1 unit of iTaq
(BIO-RAD), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM MgC12. Reactions were run
with 2.6 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platform
with 2 ng of target DNA. Target DNA was genomic DNA made from cells
homozygous for the two SMAD7 alleles (Coreill 18562 and 18537). The "C" and
"T"
alleles (SEQ ID Nos. 239 and 240) were tested individually (homozygote) and
together
(heterozygote). Reactions were started with a soak at 95 C for 5 minutes
followed by
45 cycles of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C for 1
second]. Data
acquisition was set for multiplex mode detecting the FAM and HEX channels.
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CA 02949315 2016-11-23
10477] Results are shown in Figure 30. It is clear that the FAM-labeled "C"
probe
detected the presence of the "C" target DNA but not the "T" target DNA and
that the
HEX "T" probe detected the presence of the "T" target DNA but not the "C"
target
DNA. Thus FQ cleavable primers can be used in multiplex formats to distinguish
SNPs.
EXAMPLE 21 Use of fluorescence-quenched cleavable primers in the
primer-probe assay
10478] We previously described a method of detecting nucleic acid samples
using
fluorescence quenched primers comprising two distinct but linked elements, a
Reporter
Domain positioned towards the 5'-end and a Primer Domain, positioned at the 3'-
end
of the nucleic acid molecule (US patent application US 2009/0068643). The
Primer
Domain is complementary to and will bind to a target nucleic acid under
conditions
employed in PCR. It is capable of priming DNA synthesis using the
complementary
target as template, such as in PCR. The Reporter Domain comprises a sequence
which
can be complementary to the target or can be unrelated to the target nucleic
acid and
does not hybridize to the target. Furthermore the Reporter Domain includes a
detectable element, such as a fluorescent reporter dye, and a quencher. The
reporter
dye and quencher are separated by a suitable number of nucleotides such that
fluorescent signal from the reporter dye is effective suppressed by the
quencher when
the Reporter Domain is in single-stranded random coil conformation. During
PCR, the
Primer Domain will prime DNA synthesis and the FQT synthetic oligonucleotide
is
thereby incorporated into a product nucleic acid, which itself can is used as
template in
the next cycle of PCR. Upon primer extension during the next cycle of PCR, the
entire
FQT probe is converted to double-stranded form, including the Reporter Domain.
Formation of a rigid double-stranded duplex physically increases the distance
between
the fluoropohorc and the quencher, decreasing the suppression of fluorescence
emission (hence increasing fluorescent intensity). Thus conversion of the FQT
primer
to double-stranded form during PCR constitutes a detectable event. Further
increases
in fluorescent signal can be achieved by cleavage of the Reporter Domain at a
site
between the reporter dye and the quencher, such that the reporter dye and the
quencher
become physically separated and are no longer covalently linked on the same
nucleic
acid molecule. This cleavage event is dependent upon formation of double-
stranded
137
CA 02949315 2016-11-23
nucleic acid sequence so that cleavage cannot occur if the FQT primer is in
its original
single-stranded state. Suitable methods to separate reporter and quencher
include, for
example, use of a restriction endonuclease to cleave at a specific sequence in
dsDNA.
Alternatively, an RNase H2 cleavage domain can be placed between the
fluorophore
and quencher. Placement of a single ribonucleotide residue between the
fluorophorc
and the quencher would make the FQT primer a suitable substrate for RNase H2
during
PCR. The scheme for this reaction is shown in Figure 31. The present example
demonstrates use of a thermostable RNase H2 to mediate cleavage of a
fluorescence-quenched primer in a primer-probe real time PCR assay.
104791 A qPCR assay was designed for the human Drosha gene including
unmodified For and Rev primers with an internally positioned dual-labeled
probe
suitable for use in the 5'-nuclease assay. The For primer was also synthesized
as an
FQT forward primer using the same Primer Domain sequence as the unmodified For
primer and adding a Reporter Domain on the 5'-end comprising a reporter dye
(Fluorescein-dT) and a dark quencher (IBFQ) separated by 11 bases including a
centrally positioned rU base (cleavage site). Sequences arc shown below in
Table 47.
Table 47:
Name Sequence SEQ ID No.
Drosha-For 5' -ACCAACGACAAGACCAAGAG SEQ ID No. 241
Drosha-Rev 5'-TCGTGGAAAGAAGCAGACA SEQ ID No. 242
Drosha-probe 5' -FAM-ACCAAGACCTTGGCGGACCTTT-IBFQ SEQ ID No. 243
Drosha-For-FQT 5' - I BFQ- TTTCCuGGT T T ( Fl - dT ) ACCAACGACAAGACCAAGAG SEQ
ID No. 244
DNA bases are shown in uppercase. RNA bases are shown in lowercase. Fl-dT in
an
internal Fluorescein-dT modified base. IBFQ is Iowa Black FQ, a dark quencher.
The
portion of the FQT probe that is complementary to the Drosha target is
underlined (i.e.,
the Primer Domain).
104801 The above primers target the following 141 bp region of the human
Drosha
gene (RNASEN, NM 013235). Primer binding sites are underlined and the internal
probe binding site for the 5'-nuclease assay is in bold font.
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CA 02949315 2016-11-23
Drosha amplicon (SEQ ID No. 245)
ACCAACGACAAGACCAAGAGGCCTGTGGCGCT TCGCACCAAGACCTTGGCGGACCTTTTGGAATCATTT
ATTGCAGCGCTGTACATTGATAAGGATTTGGAATATGTTCATACTTTCATGAATGTCTGCTTCTTTCCA
CGA
104811 5'-Nuclease qPCR reactions were performed in 10 ill volume using 200
nM
unmodified For and Rev primers with 200 nM probe, 200 iuM of each dNTP (800
jaM
total), 1 unit of iTaq (BIO-RAD), 50 mM Tris pH 8.3, 50 mM KC1, and 3 mM
MgC12.
FQT qPCR reactions were performed in 10 1d volume using 200 nM FQT-For primer
and 200 nM unmodified Rev primer, 200 ,uM of each dNTP (800 1.1M total), 1
unit of
iTaq (hot start thermostable DNA polymerase, BIO-RAD), 50 mM Tris pH 8.3, 50
mM
KC1, and 3 mM MgC12. Reactions were run with or without 2.6 mU of Pyrococcus
abyssi RNase H2 on a Roche Lightcycler 480 platform. Reactions were run with
or
without 10 ng of cDNA made from HeLa total cellular RNA. Reactions were
started
with a soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10
seconds, 60 C
for 30 seconds, and 72 C for 1 second].
104821 Results for the 5'-nuclease qPCR reaction are shown in Figure 32A. A
positive signal was seen at cycle 26. Results for the FQT primer qPCR
reactions arc
shown in Figure 32B. A positive signal was seen at cycle 27, nearly identical
to the
5'-nuclease assay results. In this case, signal was dependent upon RNase H2
cleavage.
Thus cleavage at an internal RNA residue by RNase 112 can be used to generate
signal
from FQT primers that have a distinct fluorescence-quenched reporter domain.
EXAMPLE 22 Use of modified bases in cleavable blocked primers to improve
mismatch discrimination
[0483] We demonstrated that blocked cleavable primers can be used in qPCR
to
distinguish single base mismatches in the SYBR Green assay format in Example
13 and
in the fluorescence-quenched (FQ) assay format in Example 20. Depending upon
the
precise base mismatch and the sequence context, detectable signal for the
mismatch
target occurred from 5 to 15 cycles after detection of the perfect match
target. There
may be circumstances where greater levels of mismatch discrimination are
desired,
such as detection of a rare mutant allele in the background of predominantly
wild type
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CA 02949315 2016-11-23
cells. We demonstrate in this example that selective placement of 2'0Me RNA
modified residue within the cleavable primer can improve mismatch
discrimination.
[0484] Example 5 above demonstrated that modified bases could be compatible
with cleavage of a heteroduplex substrate by RNase H2 depending upon the type
of
modification employed and placement relative to the cleavage site. Here we
demonstrate in greater detail use of the 2'0Mc modification in blocked primers
having
a single unmodified ribonucleotide base. The following primers, shown below in
Table
48, were synthesized and used in ciPCR reactions in the SYBR Green format with
a
synthetic oligonucleotide template. Blocked cleavable primers having a single
rU
residue were synthesized either without additional modification (SEQ ID No.
134) or
with a 2'0Me base 5'- to the rU (SEQ ID No. 247) or with a 2'0Me base 3'- to
the rU
(SEQ ID No. 248). If the 2'0Me residue is positioned 5'- to the
ribonucleotide, then it
will remain in the final primer which results from cleavage by RNase H2.
Therefore a
Syn-Rev-mU primer was made specific for the synthetic template bearing a 3'-
2'0Me
U residue at the 3'-end to mimic this reaction product (SEQ ID No. 246).
Table 48:
Name Sequence SEQ ID No.
Syn-For 5' -AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 86
Syn-Rev 5'-CTGAGCTTCATGCCTTTACTGT SEQ ID No. 87
Syn-Rev-m U 5' -CTGAGCTTCATGCCTTTACTG (mU) SEQ ID No. 246
Syn-Rev-rU-C3 5f-CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 SEQ ID No. 134
Syn-Rev-mUrU-C3 5' -CTGAGCTTCATGCCTTTACTG (mil) uCCCC-SpC3 SEQ ID No. 247
Syn-Rev-rUmC-C3 5' -CTGAGCTTCATGCCTTTACTGTu (mC) CCC-SpC3 SEQ ID No. 248
DNA bases are shown in uppercase. RNA bases are shown in lowercase. 2'0Me RNA
bases are indicated as (mN).
104851 The following synthetic oligonucleotide was used as template. Primer
binding sites are underlined.
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Synthetic template, SEQ ID No. 162:
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG
[0486] PCR reactions were performed in 10 il volume using 200 nM unmodified
For primer pairwise with 200 nM of each of the different Rev primers shown
above in
Bio-Rad SYBR Green master mix. Reactions were run with or without 1.3 - 200 mU
of
Pyrococcus abys,si RNase H2 on a Roche Lightcycicr 480 platformwith no target
or 2
x 106 copies of the synthetic oligonucleotide template. Reactions were started
with a
soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60 C
for 20
seconds, and 72 C for 30 seconds]. Results are summarized in Table 49.
Table 49. Cp values of c[PCR reactions comparing blocked primers with or
without a
2'0Me base flanking a cleavable ribonucleotide.
Syn-Rev Syn-Rev-mU Syn-Rev-rU-C3
RNase SEQ ID SEQ ID No. SEQ ID No. Syn-Rev-mUrU-C3 Syn-Rev-rUmC-C3
SEQ ID No. 247 SEQ ID No.
248
H2 No. 87 246 134
None 17.8 19.8 >40 >40 >40
17.8 19.8 17.2 21.6 >40
mU
100
17.8 19.6 17.2 19.7 >40
mU
150
17.8 19.9 17.2 19.5 >40
mU
200
17.8 19.8 17.2 19.1 >40
mU
[0487] The unblocked primer with a 3'-terminal 2'0Me base (SEQ ID No. 246)
showed a 2 cycle delay relative to the unmodified primer (SEQ ID No. 87),
indicating
that the terminal 2'0Me base slightly decreased priming efficiency but
nevertheless
was functional as a PCR primer. The blocked primer containing a single rU base
(SEQ
ID No. 134) performed as expected (see Example 13) and worked well with low
concentrations of RNase H2 (data not shown). For the 2'0Me RNA containing
primers
a higher concentration of RNase H2 was needed. The primer having a 2' OMe
residue
5'- to the ribonucleotide (SEQ ID No. 247) showed good activity at 50 mU RNase
H2
and performed identically to the unblocked 2'0Me control primer (SEQ ID No.
246)
when 100 mU or higher RNasc H2 was employed. The primer having a 2'0Mc residue
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CA 02949315 2016-11-23
3'-to the ribonucleotide (SEQ ID No. 248) did not function at any level of
RNase H2
tested. The primer having a 2'0Me residue 5'- to the ribonucleotide (SEQ ID
No. 247)
was next tested in a mismatch discrimination qPCR assay.
104881 The standard configuration blocked RNase H2 cleavable primer (SEQ ID
No. 134) was compared with the 5 '-2'0Me version of this sequence (SEQ ID No.
247).
These two "Rev" primers were used with the unmodified "For" primer (SEQ ID No.
86)
together with 3 different synthetic oligonucleotide templates (originally used
in
defining mismatch discrimination potential in Example 13). These templates
provide a
perfect match control (Template SEQ ID No. 162), a T/U mismatch (Template SEQ
ID
No. 155), or a G/U mismatch (Template SEQ ID No. 176). The 3 templates
oligonucleotides are shown below with the cleavable blocked primer (SEQ ID No.
134)
aligned beneath to illustrate the regions of match vs. mismatch.
Synthetic template, SEQ ID No. 162 (A:U match):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
111111111111111111111111111
3'-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
Synthetic template, SEQ ID No. 155 (T:U mismatch):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGTACAGTAAAGGCATGAAGCTCAG-3'
1111 1111111111111111111111
3'-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
Synthetic template, SEQ ID No. 176 (G:U mismatch):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGGACAGTAAAGGCATGAAGCTCAG-3'
1111 1111111111111111111111
3'-C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
104891 PCR reactions were performed in 10 ,u1 volume using 200 nM
unmodified
For primer with 200 nM of cleavable blocked Rev primer (SEQ ID No. 134) or
5'mU
containing cleavable blocked Rev primer (SEQ ID No. 247) in Bio-Rad SYBR Green
master mix. Reactions were run with 1.3 mU (primer SEQ ID No. 134) or 100 mU
(primer SEQ ID No. 247) of Pyrococcus abyssi RNase H2. Reactions were run on a
Roche Lightcycler 480 platform with 2 x 106 copies of the different synthetic
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CA 02949315 2016-11-23
oligonucleotide templates (SEQ ID Nos. 155, 162, or 176). Reactions were
started with
a soak at 95 C for 5 minutes followed by 45 cycles of [95 C for 10 seconds, 60
C for 20
seconds, and 72 C for 30 seconds]. Results are summarized in Table 50 and are
shown
as ACp (ACp = Cp mismatch ¨ Cp match).
Table50. Cp values of qPCR reactions comparing mismatch discrimination of
blocked
primers with or without a 2'0Me base on the 5'-side of an RNA residue.
1.3 mU RNase H2 100 mU RNase H2
SEQ ID No. 134 SEQ ID No. 247
rU primer mUrU primer
Match (A:U) 0 0
Mismatch (T:U) 5.3 12.7
Mismatch (G:U) 10.9 14.4
(ACp = Cp mismatch ¨ Cp match)
104901 In both cases tested, addition of a 2'0Me residue directly 5' to the
cleavable
ribonucleotide significantly improved mismatch discrimination. The T/U
mismatch
improved from a ACp of 5.3 to 12.7 and the G/U mismatch improved from a ACp of
10.9 to 14.4. This new primer design required use of 100 mU of RNase H2
compared
with 1.3 mU (in a 10 ul assay), however the enzyme is inexpensive and the
boost in
reaction specificity was considerable. We conclude that the use of chemically
modified
residues in select positions within the cleavable primer can significantly
improve the
mismatch discrimination capability of the assay.
EXAMPLE 23 Use of double-mismatch design in cleavable blocked primers to
improve mismatch discrimination
104911 Some nucleic acid probes that are complementary to a wild type (WT)
sequence will bind to both the perfect match WT target and a mutant target
bearing a
single base mismatch with sufficiently similar affinity that the two sequences
(WT and
mutant) are not easily distinguished. While a single mismatch introduced
between the
probe and target sequence may not significantly disrupt binding to the wild
type target
(which has 1 mismatch with the probe) disrupts binding to the mutant target
(which
now has 2 mismatches with the probe). This strategy has been used to improve
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CA 02949315 2016-11-23
selectivity of hybridization based assays as well as assays dependent upon
interaction
with nucleic acid binding proteins. The present example demonstrates use of a
double-mismatch strategy to improve base discrimination with use of
cleavable-blocked primers of the present invention.
[0492] For the present study, the SMAD7 qPCR SNP discrimination assay
presented in Example 20 was employed as a model system, except that the SYBR
Green detection format was used instead of the FQ format. Blocked-cleavable
primers
were synthesized with the base mismatch in the positioned at the cleavable
ribonucleotide. Using the present probe design, any mismatch placed 5'- to the
cleavage site (RNA base) will be retained in the primer extension product and
thus will
be replicated during PCR. In order to maintain the presence of the double-
mismatch
during PCR, the new mismatch must be positioned 3'- to the cleavable RNA
residue in
the domain that is cleaved off and is not retained in daughter products. It is
desirable
that the intentionally added second mismatch not disrupt function of the
primer with a
perfect match target. It was demonstrated in Example 13 that mismatches
present in the
"+1 position" (i.e., immediately 3'- to the RNA base) can have a significant
impact
upon cleavage and functional primer efficiency. The double mismatch was
therefore
placed at the "+2 position- 3'- to the RNA base with the expectation that this
configuration would not be disruptive as a single mismatch but would be
disruptive as a
double mismatch.
[0493] Blocked-cleavable primers were designed and synthesized at this site
using
standard design features to discriminate between the "C" and "T" alleles in
the SMAD7
gene (SNP locus rs4939827). The same unmodified Rev primer was used in all
assays
(SEQ ID No. 236). The perfect match "C" allele primer is SEQ ID No. 250 and
the
perfect match "T" allele primer is SEQ ID No. 254. Next, a series of primers
were
made bearing a mutation at position +2 relative to the ribonucleotide (2 bases
3'- to the
RNA residue). It was anticipated that the identity of the base mismatch would
alter the
relative perturbation that having a mismatch at this position would introduce
into the
assay. Therefore, perfect match (wild type) and all 3 possible base mismatches
were
synthesized and studied (SEQ ID Nos. 251-253 and 255-257). Sequences are shown
below in Table 51.
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Table 51:
Name Sequence SEQ ID No.
rs4939827 Rev 5f-CTCACTCTAAACCCCAGCATT SEQ ID No. 236
rs4939827 For 5f-CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 249
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3 SEQ ID No. 250
WT
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcAAGA-SpC3 SEQ ID No. 251
CAA
rs4939827 C-For
5'-CAGCCTCATCCAAAAGAGGAAAcACGA-SpC3 SEQ ID No. 252
CAC
rs4939827 C-For
5f-CAGCCTCATCCAAAAGAGGAAAcATGA-SpC3 SEQ ID No. 253
CAT
rs4939827 T-For
5'-CAGCCTCATCCAAAAGAGGAAAuAGGA-SpC3 SEQ ID No. 254
WT
rs4939827 T-For
5f-CAGCCTCATCCAAAAGAGGAAAuAAGA-SpC3 SEQ ID No. 255
UAA
rs4939827 T-For
5'-CAGCCTCATCCAAAAGAGGAAAuACGA-SpC3 SEQ ID No. 256
UAC
rs4939827 T-For
5'-CAGCCTCATCCAAAAGAGGAAAuATGA-SpC3 SEQ ID No. 257
UAT
DNA bases are shown in uppercase. RNA bases are shown in lowercase. SpC3 is a
spacer C3 used as a 3'-blocking group. Mutations
introduced to create
double-mismatches are indicated with bold underline.
[04941 The above
primers target the following 85 bp region of the SMAD7 gene
(NM 005904). Primer binding sites are underlined and the SNP location is
highlighted
as bold italic. Primers are aligned with target in Figure 33 to help
illustrate the scheme
of the "double mutant" approach to improve SNP discrimination.
rs4939827 (SMAD7) C allele (SEQ ID No. 239)
CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
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rs4939827 (SMAD7) T allele (SEQ ID No. 240)
CAGCCTCATCCAAAAGAGGAAATAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
[0495] PCR reactions were performed in 10 111 volume using 200 nM of the
unmodified Rev primer (SEQ ID No. 236) and the series of cleavable blocked For
primers (SEQ ID Nos. 250-257) in Bio-Rad SYBR Green master mix. Reactions were
run with 2.6 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480
platform
with 2 ng of target DNA. Target DNA was genomic DNA made from cells
homozygous for the two SMAD7 alleles (Coreill 18562 and 18537). The "C" and
"T"
alleles (SEQ ID Nos. 239 and 240) were tested individually. Reactions were
started
with a soak at 95 C for 5 minutes followed by 80 cycles of [95 C for 10
seconds, 60 C
for 30 seconds, and 72 C for 1 second]. Results are shown in Table 52 below.
Table 52. Cp and ACp values of qPCR reactions comparing mismatch
discrimination of
blocked primers with or without a second mutation at position +2 relative to
the
ribonucleotide.
Cp "C" Allele Cp "T" Allele ACp
SEQ ID No. Unblocked
27.5 26.5
249 control
SEQ ID No.
rCAG (WT) 29.2 39.9 10.7
250
SEQ ID No.
rCAA 29.0 47.8 18.8
251
SEQ ID No.
rCAC 31.6 45.4 13.8
252
SEQ ID No.
rCAT 30.2 42.8 12.6
253
SEQ ID No.
rUAG (WT) 42.6 29.2 13.4
254
SEQ ID No.
rUAA 49.3 40.1 9.2
255
SEQ ID No.
rUAC 74.1 49.9 24.2
256
SEQ ID No.
rUAT 62.5 45.3 17.2
257
(ACp = Cp mismatch ¨ Cp match)
[0496] For the "C" allele, the standard design perfectly matched probe (SEQ
ID No.
250) showed amplification efficiency similar to unmodified control primers and
the
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CA 02949315 2016-11-23
mismatch discrimination was 10.7 cycles (ACp = 10.7) against the "T" target.
The
mismatch primers showed a minor decrease in detection efficiency with the "C"
allele
target (a shift of up to 2.4 cycles was observed) but mismatch discrimination
at the SNP
site increased significantly with a ACp of 18.8 cycles seen for the rCAA
primer (SEQ
ID No. 251).
104971 For the "T" allele, the standard design perfect match probe (SEQ ID
No. 254)
also showed amplification efficiency similar to unmodified control primers and
the
mismatch discrimination was 13.4 cycles (ACp = 13.4) against the "C" target.
However, unlike the "C" allele, the mismatch primers for the "T" allele showed
a large
decrease in detection efficiency with the "T" allele target. Shifts as large
as 20 cycles
were observed. Nevertheless the relative SNP discrimination was improved with
a ACp
of 24.2 cycles seen for the rUAC primer (SEQ ID No. 256). For this region of
the
SMAD7 gene, the "T" allele creates an "AT-rich" stretch at the site of the
cleavable
RNA base and this sequence has low thermal stability. The presence of a
mismatch at
the +2 position must destabilize the structure in this region much more for
the "T" allele
than the higher stability "C" allele, which would account for the observed
increase Cp
for the "T" allele probes against the "T" target. However, this shift in Cp
values does
not limit utility of the assay. Given the inherent increased specificity of
the
blocked-cleavable primers (see Example 11), there should be no problem with
routinely
extending reactions to 60-80 or more cycles. In certain settings, the
increased
discrimination power of the double-mismatch format will be of sufficient value
to
accept the lower overall reaction efficiency. In "AT-rich" regions like the
SMAD7 "T"
allele, it might also be useful to position the double mismatch at the +3
position,
removing its disruptive effects further from the cleavable ribonucleotide.
EXAMPLE 24 Identity of reaction products made by PCR amplification at SNP
sites using cleavable-blocked primers
104981 For use in PCR or any primer extension application, if a base
mismatch
(SNP site) is positioned directly at the ribonucleotide residue in blocked-
cleavable
primers, then a cleavage event that occurs 5'- to the RNA base will result in
a primer
extension product that reproduces the base variant present in the template
nucleic acid.
A cleavage event that occurs 3"- to the RNA base will result in a primer
extension
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CA 02949315 2016-11-23
product that changes the product to the RNA base present in the primer,
creating an
error that will be replicated in subsequent PCR cycles. Cleavage on the 3'-
side of the
ribonucleotide is therefore an undesired event. Given the enormous
amplification
power of PCR, even a small amount of 3'-cleavage could lead to the
accumulation of a
sizeable amount of products containing a sequence error. For example, cleavage
at a
rate of 0.1% would lead to 1 out of 1000 molecules having the -wrong" base at
the site
of the RNA residue which would then be detectable as "perfect match" in
subsequent
PCR cycles. This would equate to a 10 cycle shift (ACp = 10) in a qPCR
reaction.
Using the design parameters taught in Example 13, cycle shifts for SNP
discrimination
varied from 5-15. Thus a small amount of undesired and unsuspected 3'-cleavage
could easily account for the delayed false-positive signals seen in Example 13
during
SNP interrogation.
04991 A false positive signal in an allele-specific SNP discrimination
reaction
could arise from two sources. First, ongoing inefficient cleavage at the
"normal"
RNasc H2 cleavage site at the 5'-side of the RNA base (see Figure 3) in spite
of the
mismatch. This reaction will result in primer extension products identical to
the
starting target. Second, a false positive signal in an allele-specific SNP
discrimination
reaction could also arise from inefficient cleavage at an "abnormal" position
anywhere
on the 3 '-side of the RNA base. This reaction would produce primer extension
products identical to the primer and which would then amplify with high
efficiency
using this same primer. If the first scenario were correct, then the products
from a
reaction performed using allele "A" primer with allele "B" target should
produce
mostly allele "B" products, which would continue to amplify inefficiently with
allele
"A" primers. If the second scenario were correct, then the products from a
reaction
performed using allele "A" primer with allele "B" target should produce mostly
allele
"A" products, which would amplify efficiently with allele "A" primers.
10500] To distinguish between these possibilities, a re-amplification
experiment
was performed wherein a first round of PCR amplification was performed using a
SMAD7 "T" allele primer with SMAD7 "T" allele target DNA or with SMAD7 "C"
allele target DNA. The reaction products were diluted 108 fold and re-
amplification
was performed using the "T" vs. "C" allele primers to determine if the
identity of the
SNP base present in the reaction products changed during the first round of
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CA 02949315 2016-11-23
amplification. The SMAD7 rs4939827 allele system was employed using the
following primers and target DNAs, which are shown below in Table 53.
Table 53:
Name Sequence SEQ ID No.
rs4939827 Rev 5f-CTCACTCTAAACCCCAGCATT SEQ ID No. 236
rs4939827 For 5 -CAGCC T CAT C CAAAAGAGGAAA SEQ ID No. 249
rs4939827 C-For
-CAGCCTCATCCAAAAGAGGAAAcAGGA- SpC 3 SEQ ID No. 250
WT
rs4939827 T-For
5 -CAGCC TCATCCAAAAGAGGAAAuAGGA-SpC 3 SEQ ID No. 254
WT
DNA bases are shown in uppercase. RNA bases are shown in lowercase. SpC3 is a
spacer C3 used as a 3'-blocking group.
[0501] The above primers target the following 85 bp region of the SMAD7
gene
(NM 005904). Synthetic oligonucleotides were synthesized for use as pure
targets in
the SMAD7 system and are shown below. Primer binding sites are underlined and
the
SNP location is highlighted as bold italic.
rs4939827 (SMAD7) C allele (SEQ ID No. 239)
CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
rs4939827 (SMAD7) T allele (SEQ ID No. 240)
CAGCCTCATCCAAAAGAGGAAA TAGGACCCCAGAGCTCCCTCAGAC TCCTCAGGAAACACAGACAATGC
TGGGGTTTAGAGTGAG
[0502] PCR reactions were performed in 10 pl volume using 200 nM of the
unmodified Rev primer (SEQ ID No. 236) and the "T" allele cleavable blocked
For
primer (SEQ ID No. 254) in Bio-Rad SYBR Green master mix. Reactions were run
with 2.6 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler(R) 480
platform
with 6.6 x 105 copies of synthetic oligonucleotide target SMAD7 "C" allele
(SEQ ID
No. 239) or SMAD7 "T" allele (SEQ ID No. 249). Reactions were started with a
soak
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CA 02949315 2016-11-23
at 95 C for 5 minutes followed by 80 cycles of [95 C for 10 seconds, 60 C for
30
seconds, and 72 C for 1 second]. Results of qPCR amplifications done at this
SNP site
are shown in Table 54 below.
Table 54. Cp and ACp values of qPCR reactions showing mismatch discrimination
of
cleavable-blocked primers at a SMAD7 C/T allele.
Cp for: Cp for:
"T" Target "C" Target ACp
SEQ ID No. 240 SEQ ID No. 239
Primer
rs4939827 T-For WT 32.5 18.9 13.6
SEQ ID No. 254
[0503] The "T" allele primer performed similar to pervious results showing
a ACp
of 13.6 between reactions run using the match "T" allele target DNA and the
mismatch
"C" allele target DNA.
[0504] This experiment was repeated using a 108 dilution of the reaction
products
from the above PCR amplifications as target DNA. If cleavage at the mismatch
site
occurred at the expected position 5'- to the ribonucleotide, then the reaction
products
should remain "true" and "T" allele product would be made from input "T"
allele
template and "C" allele product would be made for input "C" allele template.
However,
if any appreciable cleavage occurred 3'- to the ribonucleotide, then the
reaction
products should be converted to the sequence of the primer at the SNP site. In
this case,
a "T" allele product would be made from a "C" allele target.
[0505] PCR reactions were performed in 10 pl volume using 200 nM of the
unmodified Rev primer (SEQ ID No. 236) and the "T" allele cleavable blocked
For
primer (SEQ ID No. 254) or the "C" allele cleavable blocked For primer (SEQ ID
No.
250) in Bio-Rad SYBR Green master mix. Reactions were run with 2.6 mU of
Pyrococcus abyssi RNase H2 on a Roche Lightcyclei 480 platform. Input target
DNA
was a 108 dilution of the reaction products shown in Table 54 above. Reactions
were
started with a soak at 95 C for 5 minutes followed by 45 cycles of [95 C for
10 seconds,
60 C for 30 seconds, and 72 C for 1 second]. Results of qPCR amplifications
done at
this SNP site arc shown in Table 55 below.
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CA 02949315 2016-11-23
Table 55. Cp and ACp values of qPCR reactions showing mismatch discrimination
of
cleavable-blocked primers at a SMAD7 C/T
Cp for: Cp for:
"T" Target amplified "C" Target amplified ACp
by "T" primer by "T" primer
Primer
rs4939827 T-For WT 27.7 29.2 1.5
SEQ ID No. 254
Primer
rs4939827 C-For WT 38.6 38.5 0.1
SEQ ID No. 250
105061 The reactions products previously made (Table 54) using the "T"
allele
primer with both the "T" allele target and the "C" allele target now show
nearly
identical amplification efficiency using the "T" allele primer whereas
previously a ACp
of 13.6 was observed between the two different starting target DNAs. This is
most
consistent with the product nucleic acids having similar sequence, i.e., both
are now
predominantly "T" allele. Consistent with this hypothesis, both of these
samples now
show similar delayed Cp using the "C" allele primer. Thus it appears that the
product
from the "T" allele primer amplification using the "C" allele target was
largely
converted to "T" allele, consistent with that product originating with a
primer cleavage
event occurring 3'- to the ribonucleotide base. The reaction products from the
original
amplification using the "T- allele primer (Table 54) were subcloned and DNA
sequence determined. All clones identified had the "T" allele present, whether
the
starting template was the "T" allele or the "C" allele, adding further support
to this
conclusion.
EXAMPLE 25 Use of phosphorothioate modified internucleotide linkages in
cleavable blocked primers to improve mismatch discrimination
105071 The results form Example 24 indicates that PCR performed with a
mismatched primer/target combination can produce a product with sequence
matching
the primer instead of the target. The most likely scenario that would result
in this kind
of product starts with cleavage of the mismatched primer at a position 3'- to
the
ribonucleotide residue. Use of chemical modifications that prevent unwanted
cleavage
in this domain of the primer may improve performance of the cleavable-blocked
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CA 02949315 2016-11-23
primers especially in SNP discrimination. The following primers, as shown in
below in
Table 56, were synthesized with nuclease-resistant phosphorothioate (PS)
modified
internucleotide linkages placed at positions 3'- to the ribonucleotide as
indicated. It
was established in Example 15 that placement of a PS bond at the 3'-linkage
directly at
the RNA base can decrease cleavage efficiency. This modification survey
therefore
focused on the DNA linkages further 3'- to this site. The synthetic amplicon
system
previously used in Example 13 was employed.
Table 56:
Name Sequence SEQ ID No.
Syn-For 5'-AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 86
Syn-Rev-rU-C3 5'-CTGAGCTTCATGCCTTTACTGTuCCCC-SpC3 SEQ ID No. 134
Syn-Rev-rU-
5'-CTGAGCTTCATGCCTTTACTGTuC*CCC-SpC3 SEQ ID No. 257
C*CCC-C3
Syn-Rev-rU-
5f-CTGAGCTTCATGCCTTTACTGTuCC*CC-SpC3 SEQ ID No. 258
CC*CC-C3
Syn-Rev-rU-
5'-CTGAGCTTCATGCCTTTACTGTuCCC*C-SpC3 SEQ ID No. 259
CCC*C-C3
,
CSyn-Rev-rU-
C-C3 5f-CTGAGCTTCATGCCTTTACTGTuC*C*C*C-SpC3 SEQ ID No. 260
C*C**
DNA bases are shown in uppercase. RNA bases arc shown in lowercase.
"*" indicates a phosphorothioatc (PS) modified internucleotide linkage.
105081 The standard configuration blocked RNase H2 cleavable primer (SEQ ID
No. 134) was compared with PS-modified versions of this sequence (SEQ ID Nos.
257-60). This set of "Rev" primers were used with the unmodified "For" primer
(SEQ
ID No. 86) together with two different synthetic oligonucleotide templates
(originally
used in defining mismatch discrimination potential in Example 13). These
templates
provide a perfect match control (Template SEQ ID No. 162) and a T/U mismatch
(Template SEQ ID No. 155). The two templates and oligonucleotides are shown
below
with the cleavable blocked primer (SEQ ID No. 134) aligned beneath to
illustrate the
regions of match vs. mismatch.
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CA 02949315 2016-11-23
Synthetic template, SEQ ID No. 162 (A:U match):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
111111111111111111111111111
3' -C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
Synthetic template, SEQ ID No. 155 (T:U mismatch):
AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTTGGCCTCAGAAGTAGTGGCCAGCT
GTGTGTCGGGGTACAGTAAAGGCATGAAGCTCAG- 3'
1111 1111111111111111111111
3' -C3-CCCCuTGTCATTTCCGTACTTCGAGTC-5'
105091 PCR reactions were performed in 10 },11 volume using 200 nM of the
unmodified For primer (SEQ ID No. 86) and the different cleavable blocked Rev
primers shown above (SEQ ID Nos. 134, 257-260) in Bio-Rad SYBR Green master
mix. Reactions were run with 1.3 mU of Pyrococcus abyssi RNasc H2 on a Roche
Lightcyclee' 480 platform. Input target DNA was 2 x 106 copies of the
synthetic target
sequences shown above (SEQ ID Nos. 155 and 162). Reactions were started with
an
incubation at 95 C for 5 minutes followed by 45 cycles of [95 C for 10
seconds, 60 C
for 30 seconds, and 72 C for 1 second]. Results of qPCR amplifications done at
this
SNP site are shown in Table 57 below.
Table 57. Cp values of qPCR reactions comparing mismatch discrimination of
blocked
primers with or without PS linkages 3'- to the cleavable ribonucleotide.
Match Target Mismatch Target
(A: U) (T: ACp
SEQ ID No. 162 SEQ ID No. 155
SEQ ID No. 134
18.5 26.2 7.7
CCCC Primer
SEQ ID No. 257
19.5 31.9 12.4
C*CCC Primer
SEQ ID No. 258
18.2 26.7 8.5
CC*CC Primer
SEQ ID No. 259
18.4 26.3 7.9
CCC*C Primer
SEQ ID No. 260
18.5 29.1 10.6
C*C*C*C Primer
(ACp = Cp mismatch ¨ Cp match
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[0510] Placement of a PS modified linkage at the 3' "+1" position (rUC*CCC)
led
to almost a 5 cycle improvement in SNP discrimination in this assay (SEQ ID
No. 260
vs. 134), demonstrating that increasing nuclease stability in the domain 3'-
to the
ribonucleotide can significantly improve assay performance. Modification o the
linkages further 3' from the ribonucleotidc had minimal impact. Modification
of all of
the linkages in this area (rUC*C*C*C, SEQ ID No. 260) also showed benefit,
improving relative SNP discrimination by 3 cycles, but unexpectedly showed
less
benefit than using just a single modification at the 3'+1 linkage. This may
relate to the
lowered binding affinity Tm that also results from the PS modification.
[0511] Thus, adding nuclease resistant modifications at the linkages 3'- to
the
cleavable ribonucleotide can increase SNP discrimination for the RNase 112
mediated
cleavable-blocked primer PCR assay. Typically, only one of the two
stereoisomers at a
PS linkage (the Rp or Sp isomer) confers benefit. Improved activity might
therefore be
realized by isolation a chirally pure PS compound here, as was demonstrated in
Example 15. Other nuclease resistant modifications may be suitable in this
area, such
as the non-chiral phosphorodithioatc linkage, the methyl phosphonate linkage,
the
phosphoramidate linkage, a boranophosphate linkage, and abasic residues such
as a C3
spacer to name a few.
EXAMPLE 26 Use of cleavable primers having an unblocked 3'-hydroxyl in a
qPCR assay
[0512] In the above Examples, a blocking group was placed at the 3'-end of
the
primer to prevent primer extension from occurring prior to RNase H2 cleavage.
For
certain primer designs and applications, it may not be necessary or even
desirable to
employ a 3'-blocking group. We have previously described a method of nucleic
acid
amplification termed polynomial amplification that employs primers that are
chemically modified in ways that block template function while retaining
primer
function. A variety of groups can be used for this purpose, including internal
C3
spacers and internal 2'0Me RNA bases. Using nested primers, high specificity
is
achieved and amplification power is dependent upon the number of nested
primers
employed, with amplification occurring according to a polynomial expansion
instead of
the exponential amplification seen in PCR (see US Patent 7,112,406 and pending
US
Patent applications 2005/0255486 and 2008/0038724). Combining elements of the
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CA 02949315 2016-11-23
polynomial amplification primers with an RNase H2 cleavable domain of the
present
invention results in a novel primer design that has an unblocked 3'-hydroxyl
and is
capable of supporting primer extension yet cannot support PCR. Upon cleavage,
the
template blocking groups are removed and primer function for use in PCR is
restored.
The present example demonstrates use of cleavable template-blocked primers
having a
3'-hydroxyl in VCR.
105131 The following primers, as shown below in Table 58, were synthesized
for
use with the artificial synthetic amplicon used in previous Examples.
Table 58:
SEQ ID
Name Sequence
No.
Syn-For 5 -AGCTCTGCCCAAAGATTACCCTG SEQ ID
No. 86
Syn-Rev 5 -CTGAGCTTCATGCCTTTACTGT SEQ ID
No. 87
SEQ ID
Syn-For-rA-C3 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC-SpC3
No. 261
SEQ ID
Syn-For-rA-iC3-D1 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3 ) A
No. 262
SEQ ID
Syn-For-rA-iC3-D2 5 -AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3 ) AG
No. 263
Syn-For-rA-iC3-D4 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3) AGTG SEQ ID
No. 264
SEQ ID
Syn-For-rA-iC3-D5 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3 ) AGTGG
No. 265
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3'-end.
[0514] The synthetic amplicon oligonucleotide template (SEQ ID No. 162) is
shown below with the unmodified and various modified cleavable For primers
shown
aligned above and the unmodified Rev primer aligned below. DNA bases are
uppercase, RNA bases are lowercase, and "x" indicates a Spacer-C3 group.
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5'AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 86 unmodified
5'AGCTCTGCCCAAAGATTACCCTGaCAGC-x SEQ ID No. 261 3'-block
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxA SEQ ID No. 262 Int D1
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAG SEQ ID No. 263 Int D2
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTG SEQ ID No. 264 Int D4
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTGG SEQ ID No. 265 Int D5
1111111111111111111111111111 11111
5'AGCTCTGCCCAAAGATTACCCTGACAGCTAAGTGGCAGTGGAAGTIGGCCTCAGAAGTAGTGGCCAG
CTGTGTGTCGGGGAACAGTAAAGGCATGAAGCTCAG-3'
1111111111111111111111
TGTCATTTCCGTACTTCGAGTC-5' SEQ ID No. 87
[0515] PCR reactions were performed in 10 1 volume using 200 nM of the
individual For primers (SEQ ID Nos. 86, 261-65) and the unmodified Rev primer
(SEQ
ID No. 87) in Bio-Rad SYBR Green master mix. Reactions were run with or
without
1.3 mU of Pyrococcus abyssi RNase H2 on a Roche Lightcycler 480 platform.
Input
target DNA was 2 x 106 copies of the synthetic target shown above (SEQ ID No.
162).
Reactions were started with an incubation at 95 C for 5 minutes followed by 60
cycles
of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C for 1 second]. Results
of qPCR
amplifications are shown in Table 59 below.
Table 59. Cp values of qPCR reactions comparing performance of cleavable
primers
having a 3'-blocking group vs. cleavable primers having internal template
blocking
groups.
Without + 1.3 mU
RNase H2 RNase 112
Unblocked For SEQ ID No. 86
17.0 17.2
Unblocked Rev SEQ ID No. 87
3'-blocked For SEQ ID No. 261
>60 17.1
Unblocked Rev SEQ ID No. 87
It-blocked For SEQ ID No. 262 (DI)
>60 17.1
Unblocked Rev SEQ ID No. 87
Int-blocked For SEQ ID No. 263 (D2)
>60 17.1
Unblocked Rev SEQ ID No. 87
Int-blocked For SEQ ID No. 264 (D4)
>60 17.1
Unblocked Rev SEQ ID No. 87
Int-blocked For SEQ ID No. 265 (D5)
>60 17.9
Unblocked Rev SEQ ID No. 87
[0516] The unblocked primers gave detectable signal at around cycle 17 in
this
assay system. Using the unblocked Rev primer with the 3'-blocked For primer,
no
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CA 02949315 2016-11-23
signal was detected within the 60 cycle PCR run without RNase H2, however with
RNase H2 a similar cycle detection time of around 17 was seen. The internally
blocked
For primers that had a free 3'-hydroxyl group behaved identically to the 3'-
modified
primer. In spite of the unblocked 3'-hydroxyl, primer cleavage with RNase H2
was
required for function in PCR, presumably due to the loss of template function
imposed
by the internal C3 spacers. C3 spacers placed near the 3'-end may also inhibit
primer
extension to a certain degree. No signal was detected in the absence of RNase
H2; with
RNase H2, cleavage and amplification proceeded normally.
105171 This example demonstrates that cleavable primers do not need to be
modified at the 3'-terminal residue to function in a cleavable-primer PCR
assay and
that primers having internal modifications that disrupt template function can
perform
equally well. Given the significance of this finding to primer design, a
similar
experiment was performed using an endogenous human gene target using human
genomic DNA to ensure that these results could be generalized.
105181 The following primers, as shown below in Table 60, were synthesized
based
upon the human SMAD7 gene used in previous Examples, using only the "C"
allele.
Table 60:
Name Sequence SEQ ID No.
rs4939827 Rev 5' -CTCACTCTAAACCCCAGCATT SEQ ID No. 236
rs4939827 For 5' -CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 249
rs4939827
5'-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3 SEQ ID No. 250
C-For-C3
rs4939827
5'-CAGCCTCATCCAAAAGAGGAAAcAGGA(SpC3-SpC3)C SEQ ID No. 266
C-For-iC3-D1
rs4939827
5'-CAGCCTCATCCAA2AGAGGAAAcAGGA(SpC3-SpC3)CC SEQ ID No. 267
C-For-iC3-D2
rs4939827
5f-CAGCCTCATCCAAAAGAGGAAAcAGGA(SpC3-SpC3)CCAG SEQ ID No. 268
C-For-i C3-D4
rs4939827
5'-CAGCCTCATCCAAAAGAGGAAAcAGGA(SpC3-SpC3)CCAGA SEQ ID No. 269
C-For-iC3-D5
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
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CA 02949315 2016-11-23
SpC3 is a Spacer C3 group, positioned either internal within the primer
(template block)
or at the 3 '-end (primer block).
[0519] The SMAD7
amplicon sequence (SEQ ID No. 239) is shown below with the
unmodified and various modified cleavable For primers shown aligned above. DNA
bases are uppercase, RNA bases are lowercase, and "x" indicates a Spacer-C3
group.
5'CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 249 unmodified
5'CAGCCTCATCCAAAAGAGGAAAcAGGA-x SEQ ID No. 250 3'-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxC SEQ ID No. 266 It-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxCC SEQ ID No. 267 It-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxCCAG SEQ ID No. 268 It-block
5'CAGCCTCATCCAAAAGAGGAAAcAGGAxxCCAGA SEQ ID No. 269 It-block
111111111111111111111111111 11111
5'CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAAT
GCTGGGGTTTAGAGTGAG-3'
105201 PCR reactions
were performed in 10 1 volume using 200 nM of the
individual For primers (SEQ ID Nos. 249-50, 266-69) and the unmodified Rev
primer
(SEQ ID No. 236) in Bio-Rad SYBR Green master mix. Reactions were run with or
without 2.6 mU ofPyrococcus abyssi RNase H2 on a Roche Lightcycler 480
platform.
Input target DNA was 2 ng of genomic DNA from a human cell line (Coreill
18562,
SMAD7 "C" allele). Reactions were started with an incubation at 95 C for 5
minutes
followed by 60 cycles of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C
for 1
second]. Results of qPCR amplifications are shown in Table 61 below.
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CA 02949315 2016-11-23
Table 61. Cp values of qPCR reactions comparing performance of cleavable
primers
having a 3'-blocking group vs. cleavable primers having internal template
blocking
groups.
Without + 2.6 mU
RNase H2 RNase H2
Unblocked For SEQ ID No. 249
25.8 25.5
Unblocked Rev SEQ ID No. 236
3'-blocked For SEQ ID No. 250
>60 26.3
Unblocked Rev SEQ ID No. 236
Int-blocked For SEQ ID No. 266 (DI)
>60 26.3
Unblocked Rev SEQ ID No. 236
Int-blocked For SEQ ID No. 267 (D2)
>60 26.2
Unblocked Rev SEQ ID No. 236
It-blocked For SEQ ID No. 268 (D4)
>60 26.2
Unblocked Rev SEQ ID No. 236
Int-blocked For SEQ ID No. 269 (D5)
>60 26.7
Unblocked Rev SEQ ID No. 236
105211 The unblocked primers gave detectable signal around cycle 26 in this
assay
system using human genomic DNA. Using the unblocked Rev primer with the
3'-blocked For primer, no signal was detected within the 60 cycle PCR run
without
RNase H2, however with RNase H2 a similar cycle detection time of around 26
was
seen. All of the internally blocked For primers that had a free 3'-hydroxyl
group
behaved identically to the 3'-modified primer. No signal was detected in the
absence of
RNase H2; with RNase H2, cleavage and amplification proceeded normally with
detection occurring around 26 cycles.
105221 This example further demonstrates that cleavable primers do not need
to be
modified at the 3'-end to function in the cleavable primer qPCR assay. Primers
having
internal modifications that disrupt template function still require a primer
cleavage
event to function as primers in the assay. When cleavage is done using RNase
H2 at an
internal cleavable residue, like a single RNA base, amplification efficiency
is identical
to that seen using unmodified primers. This novel version of template-blocked
cleavable primers can be employed to perform PCR in complex nucleic acid
samples
like human genomic DNA.
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EXAMPLE 27 Cleavable primers with internal template blocking groups and a
3'-hydroxyl can prime DNA synthesis
105231 The cleavable template-blocked primers disclosed in Example 26 have
an
unblocked 3'-hydroxyl group that should permit the oligonucleotides to
function as
primers in linear primer extension reactions but the internal template-
blocking groups
prevent function in PCR as most of the primer cannot be replicated.
Consequently, no
primer binding site exists in the daughter products. Cleavage of the primer by
RNase
H2 removes the domain containing the template-blocking groups and restores
normal
primer function. The present example demonstrates that these compositions can
function to prime DNA synthesis.
105241 The following primers shown below in Table 62 were employed to
perform
linear primer extension reactions using the artificial synthetic amplicon
system used in
previous Examples.
Table 62:
SEQ ID
Name Sequence
No.
Syn-For 5' -AGCTCTGCCCAAAGATTACCCTG SEQ ID
No. 86
SEQ ID
Syn-For-rA-C3 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC-SpC3
No. 261
Syn-For-rA-iC3-D I 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC ( SpC3-SpC3 ) A SEQ ID
No. 262
SEQ ID
Syn-For-rA-iC3-D2 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3) AG
No. 263
Syn-For-rA-iC3-D4 -
AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3) AGTG SEQ ID
No. 264
SEQ ID
Syn-For-rA-iC3-D5 5' -AGCTCTGCCCAAAGATTACCCTGaCAGC (SpC3-SpC3) AGTGG
No. 265
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3'-end.
105251 A newly synthesized 103mer oligonucleotide template was made which
was
complementary to the Syn-For primers above (SEQ ID No. 270), which is shown
below
with the unmodified and various modified cleavable For primers aligned above.
DNA
bases are uppercase, RNA bases are lowercase, and "x" indicates a Spacer-C3
group.
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5'AGCTCTGCCCAAAGATTACCCTG SEQ ID No. 86 unmodified
5'AGCTCTGCCCA7AGATTACCCTGaCAGC-x SEQ ID No. 261 3'-block
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxA SEQ ID No. 262 Int D1
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAG SEQ ID No. 263 Int D2
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTG SEQ ID No. 264 Int D4
5'AGCTCTGCCCAAAGATTACCCTGaCAGCxxAGTGG SEQ ID No. 265 Int D5
1111111111111111111111111111 11111
3'TCGAGACGGGTTTCTAATGGGACTGTCGATTCACCGTCACCTTCAACCGGAGTCTTCATCACCGGTC
GACACACAGCCCCTTGTCATTTCCGTACTTCGAGTC-5'
105261 The six For
primers shown above were radiolabeled with 32P as described
above. Primer extension reactions were performed in a 20 pt volume using 0.8 U
iTaq
polymerase (Bio-Rad), 800 uM dNTPs, 3 mM MgC12, in lx iTaq buffer (20 mM Tris
pH 8.4, 50 mM KC1) and 2 nM primer and template (40 fmole of each
oligonucleotide
in the 20 ,uL reaction). Reactions were started with an incubation at 95 C for
5 minutes
followed by 35 cycles of [95 C for 10 seconds, 60 C for 30 seconds, and 72 C
for 1
second] on an MJ Research FTC-100 thermal cycler. Reactions were stopped with
the
addition of cold EDTA containing formamide gel loading buffer. Reaction
products
were separated using denaturing 7M urea, 15% polyacrylamide gel
electrophoresis
(PAGE) and visualized using a Packard CycloneTm Storage Phosphor System
(phosphorimager). The relative intensity of each band was quantified as above
and
results plotted as a fraction of total radioactive material present in the
band representing
the primer extension product. Results are shown in Figure 34.
105271 Under these
reaction conditions, 61% of the control unblocked primer (SEQ
ID No. 86) was converted into a higher molecular weight primer extension
product. As
expected, the 3'-end blocked cleavable primer (SEQ ID No. 261) did not show
any
primer extension product. Similarly, the D1 and D2 cleavable primers with
internal C3
groups and a 3'-hydroxyl (SEQ ID Nos. 262-3) also did not support primer
extension.
The cleavable primers having a slightly longer terminal DNA domains (the D4
and D5
sequences, SEQ ID Nos. 264-5) did support primer extension with the D4 showing
47%
conversion and the D5 showing 60% conversion of the primer into an extension
product,
a reaction efficiency identical to the unmodified control primer. Thus when
internal C3
spacers are placed very near the 3'-end both priming and template function are
disrupted. When placed more than 4 residues from the 3 '-end only template
function is
blocked.
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EXAMPLE 28 Use of cleavable primers with internal template blocking groups
and a 3'-hydroxyl to improve mismatch discrimination
105281 Example 24 demonstrated that cleavage of an RNA-containing primer on
the 3'-side of the RNA base by RNasc H2 is an undesired event that can
contribute to
late arising false positive signals in a qPCR SNP discrimination assay.
Example 25
demonstrated that modifications which confer nuclease resistance to this
domain can
improve SNP discrimination. The novel compositions described in examples 26
and 27
place internal C3 groups on the 3'-side of the cleavable ribonucleotide which
disrupts
template function of the primer in a domain that is removed by RNase H2
cleavage.
This example demonstrates that positioning the C3 spacer groups close to the
RNA
base improves performance of the cleavable primer in SNP discrimination using
a
format that leaves the probe "unblocked", having an unmodified 3'-hydroxyl.
105291 The following primers, as shown below in Table 63, were synthesized
for
the human SMAD7 gene similar to previous Examples. Primers were made specific
for
the "C" allele and were tested on both "C" allele and "T" allele genomic DNA
targets.
Table 63:
Name Sequence SEQ ID No.
SEQ ID No.
rs4939827 Rev 5'-CTCACTCTAAACCCCAGCATT
236
SEQ ID No.
rs4939827 For 5'-CAGCCTCATCCAAAAGAGGAAA
249
rs4939827 SEQ ID No.
5'-CAGCCTCATCCAAAAGAGGAAAcAGGA-SpC3
C-For-C3 250
rs4939827 SEQ ID No.
5'-CAGCCTCATCCAAAAGAGGAAAcA(SpC3-SpC3)A
C-For-A(C3C3)A 271
DNA bases are shown in uppercase. RNA bases are shown in lowercase.
SpC3 is a Spacer C3 group, positioned either internal within the primer or at
the 3'-end.
10530] The SMAD7 amplicon sequence (SEQ ID No. 239, "C" target) is shown
below with the unmodified and two modified cleavable For primers aligned above
it.
DNA bases are uppercase, RNA bases are lowercase, and "x" indicates a Spacer-
C3
group. The site of the rs4939827 C/T SNP is indicated with bold underline.
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5'CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 249 unmodified
5'CAGCCTCATCCAAAAGAGGAAAcAGGA-x SEQ ID No. 250 3'-block
5'CAGCCTCATCCAAAAGAGGAAAcAxxA SEQ ID No. 271 It-block
111111111111111111111111 1
5'CAGCCTCATCCAAAAGAGGAAACAGGACCCCAGAGCTCCCTCAGACTCCTCAGGAAACACAGACAAT
GCTGGGGTTTAGAGTGAG-3'
105311 The same primers are aligned with the mismatch SMAD7 amplicon
sequence (SEQ ID No. 240, "T" target).
5'CAGCCTCATCCAAAAGAGGAAA SEQ ID No. 249 unmodified
5'CAGCCTCATCCAAAAGAGGAAAcAGGA-x SEQ ID No. 250 3'-block
5'CAGCCTCATCCAAAAGAGGAAAcAxxA SEQ ID No. 271 It-block
1111111111111111111111 1 1
5'CAGCCTCATCCAAAAGAGGAAATAGGACCCCAGAGCTCCCTCAGAGTCCTCAGGAAACACAGACAAT
GCTGGGGTTTAGAGTGAG-3'
[0532] PCR reactions were performed in 10 pi volume using 200 nM of the
individual For primers (SEQ ID Nos. 249-50, 266-69) and the unmodified Rev
primer
(SEQ ID No. 236) in Bio-Rad SYBR Green master mix. Reactions were run with or
without 2.6 mU of PyrOCOCCIIS abyswi RNase H2 on a Roche Lightcycler 480
platform.
Input target DNA was 2 ng of genomic DNA from human cell lines homozygous for
the
SMAD7 "C" and "T" alleles (Coreill 18562 and 18537). Reactions were started
with
an incubation at 95 C for 5 minutes followed by 75 cycles of [95 C for 10
seconds,
60 C for 30 seconds, and 72 C for 1 second]. Results of qPCR amplifications
are
shown in Table 64 below.
Table 64. Cp values of qPCR reactions comparing performance of cleavable
primers
having a 3'-blocking group vs. cleavable primers having internal template
blocking
groups in a SNP discrimination assay.
Unmodified Control 3'-C3 Blocked Int-C3, 3'-unblocked
SEQ ID No. 249 SEQ ID No. 250 SEQ ID No. 271
4v-p, ¶r,
ACp
Allele Allele ACp Allele Allele ACp Allele Allele
No
27.5 25.9 >75 >75 >75 >75
RNaseH
2.6 mU
27.3 26.1 27.8 37.8 10.0 41.8 68.4 26.6
RNaseH
mU
27.1 25.8 27.0 40.0 13.0 29.9 53.7 23.8
RNaseH
50 mU
27.1 26.0 27.0 28.5 1.5 27.6 53.3 25.7
RNaseH
200 mU
27.1 25.8 27.0 26.1 27.5 41.4 13.9
RNaseH
(ACp = Cp mismatch "T" - Cp match "C")
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105331 The unmodified primers are designed to be non-discriminatory and
amplified both alleles with similar efficiency, producing a detectable signal
at around
26-27 cycles. Both cleavable primers were dependent upon RNase H2 for function
and
did not produce any detectable signal for either allele in the absence of
cleaving enzyme.
Using low amounts of RNase H2 (2.6 - 10 mU), the 3'-blocked cleavable primer
(SEQ
ID No. 250) produced detectable signal around cycle 27 for the match "C"
allele and
showed a delayed Cp of 38-40 cycles for the mismatch "T" allele (ACp of 10-
13).
Using higher amounts of RNase 112, specificity was lost and both alleles
amplified with
similar efficiency. The cleavable primer having two C3 spacers 3'- to the
ribonucleotide (SEQ ID No. 271) required higher levels of RNase H2 for
efficient
cleavage/priming and showed delayed Cp values even for the perfect match "C"
allele
using 2.6 and 10 mU of enzyme. It is not surprising that modifications of this
kind near
the RNA cleavable site require higher amounts of enzyme. Example 22
demonstrated
that placing a 2'0Me modification adjacent to the ribonucleotide required 100
mU of
RNase H2 to achieve full activity. Using higher amounts of enzyme resulted in
efficient cleavage and positive signal was detected at -27 cycles using 50 or
200 mU of
RNase 112. Importantly, SNP discrimination was markedly improved using this
primer
design, with the ACp for the "T" allele being around 25 cycles using RNase H2
in the
concentration range of 2.6-50 mU. Mismatch discrimination decreased when using
200
mU of the enzyme; however, SNP discrimination was still almost at a 14 cycle
ACp.
Optimal enzyme concentration was 50 mU, at which point priming efficiency was
similar to unmodified primers and SNP discrimination showed a 25.7 cycle ACp.
105341 Therefore the present cleavable primer design with two internal C3
spacer
groups near the ribonucleotide and an unblocked 3'-hydroxyl, "RDxxD", showed
significantly improved mismatch discrimination over the original primer
design,
"RDDDD-x" (where R = RNA base, D = DNA base, and x = C3 spacer). Related
designs, such as "RDDxxD" or "RDxxDD", may show similarly improved function
and small optimizations in design may be beneficial depending upon the precise
sequence context of the SNP of interest. Utilizing chemical modifying groups
like the
C3 spacer that disrupt template function but leave the 3'-hydroxyl unmodified
can
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enhance the specificity of cleavage at the ribonucleotide by RNase H2 and
improve
SNP discrimination.
EXAMPLE 29 Use of RNase H2-cleavable ligation probes in DNA sequencing
methods
[0535] The previous Examples described the use of RNase H2 cleavable
oligonucleotide compositions for applications as primers where the cleavable
oligonucleotide primes a DNA synthesis reaction. Applications disclosed in the
above
Examples include both end-point and real time PCR in several different
detection
formats. Example 8 showed use of cleavable primers in a DNA sequencing
application
using the Sanger sequencing method with DNA polymerase and dideoxynueleotide
terminators; in this case the RNase H2-cleavable oligonucleotide also
functioned as a
primer. RNase H2-cleavable oligonucleotides can also be used in ligation
format
assays as well. One such application is DNA sequencing using cleavable
ligation
probes. The current Example demonstrates use of RNase H2-cleavable ligation
probes
in a format suitable for use in DNA sequencing.
[0536] The use of ligation probes to sequentially interrogate the identity
of bases in
an unknown nucleic acid sequence (i.e., DNA sequencing) has been described
(see
Patents US 5,750,341 and US 6,306,597 and US application 2008/0003571). The
basic
scheme for sequencing in the 5' to 3' direction by ligation begins with a
nucleic acid
acceptor molecule hybridized to an unknown nucleic acid sequence. A series of
base
interrogation probes are hybridized to this sequence which have a known fixed
DNA
base at the 5'-end followed by random bases or universal bases to permit
stable nucleic
acid hybridization of the probe to the target nucleic acid of unknown
sequence.
Hybridization and subsequent ligation reactions are dependent upon perfect or
near
perfect match between the ligation probe and the target; perfect match is
required at the
site of ligation. Ligation leads to a detectable event which permits
identification of the
specific base present at the ligation site. An RNase H2 cleavable site is
contained
within the ligation probe. Following ligation the probe is cleaved by RNasc
H2,
releasing the bulk of the probe but leaving the newly identified base ligated
to the
acceptor nucleic acid sequence, which has now been elongated by one residue as
a
result of the cycle of ligation and cleavage. This series of enzymatic and
chemical
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events is repeated through multiple cycles of ligation, base identification,
and cleavage
and the unknown nucleic acid sequence is thereby determined.
[05371 While the
patent references cited above teach methods for sequencing by
ligation, the methods suggested therein to achieve cleavage and release of the
ligation
probe permitting multiple cycles of ligation/detection are inefficient and
difficult to
perform. RNase H2-cleavable oligonucleotides using the methods of the present
invention offer an improvement over pre-existing method and permit
construction of
less costly, easier to use cleavable ligation probes for DNA sequencing. One
scheme
for DNA sequencing using RNase H2 cleavable ligation probes is shown in Figure
35.
[0538] The RNase H2
cleavable ligation probes in this method contain a fixed
known DNA base (or bases) at the 5'-end. The fixed known base(s) can be the
single
5'-most base or can include 2 or 3 or more bases towards the 5'-end. The
present
Example employs a system wherein only the single DNA base at the 5'-end of the
probe
is fixed. The synthetic oligonucleotide has a 5'-phosphate to permit enzymatic
ligation
using a DNA ligase. An activated adenylated form of the probe can also be
used. As
mentioned, the first base at the 5'-end is fixed (known). Thus four
independent probes
are needed to perform DNA sequencing, an "A" probe, a "C" probe, a "T" probe,
and a
"G" probe. Obviously more probes will be needed if the number of fixed bases
are
greater than one (for example, 16 ligation probes will be needed if the first
2 bases are
used as fixed known sequence, one for each possible dinucleotide pair). The
first base
following the fixed known DNA residue (in this case, the second base from the
5'-end)
is a residue which is cleavable by RNase H2. In the present Example, an RNA
base is
employed, however a 2'-F residue or other cleavable modified base (such as are
described in previous Examples) can also be used. The remaining bases in the
probe
will be random bases (heterogeneous mixes of the 4 DNA bases) and/or universal
bases
(such as inosinc, 5-nitroindolc, or other such groups as arc well known to
those with
skill in the art). Total length of the probe will usually be around 8-9 bases,
however
longer or shorter probe length is possible depending on the particular ligase
enzyme
employed. When using T4 DNA ligase, a length of 8 is sufficient to achieve
efficient
hybridization and enzymatic ligation. Longer probes can also be used.
[0539] Complexity of
the probe population increases according to 4N, where N =
the number of random bases employed. For example, the probe "pTn " has a
166
CA 02949315 2016-11-23
fixed "T" base at the 5'-end, a single "n" RNA base, and 6 "N" DNA bases,
totaling 7
random residues (p = phosphate, n = RNA, N = DNA). This presents a complexity
of 47
molecules (16,384) in the population. The complexity of the probe can be
decreased by
substituting universal base groups for random N bases. This is particularly
effective
towards the 3'-end. For example, using 3 inosine residues would convert the
above
probe to "pTnNNN111" (as before, with 1 = inosine). This probe has a
complexity of 44
molecules (256). It will require a significantly lower mass input of ligation
probe to
achieve 100% ligation with a probe having a complexity of 256 than one having
a
complexity of 16,384. Use of one or more universal bases is generally
preferred.
Finally, the ligation probe has a dye molecule at or near the 3'-end to
provide a
detectable signal that can be resolved following ligation. The 3'-modifying
group also
serves to block ligation at the 3'-end so that the ligation probe itself
cannot serve as an
acceptor nucleic acid.
[0540] Use of RNase H2 cleavable ligation probes of this design in DNA
sequencing is shown schematically in Figure 35. A universal primer or acceptor
nucleic acid is hybridized to the unknown nucleic acid. Attachment of a
universal
adaptor sequence on the end of the unknown sequence may be required to permit
hybridization of the acceptor molecule, and this strategy permits use of the
same
acceptor nucleic acid for all reactions. The acceptor nucleic acid must have a
3'-hydroxyl group available for ligation. The mix of ligation probes is
introduced into
the reaction in molar excess (>256 fold excess for the 8mer inosine containing
probe
design described above) and T4 DNA ligase is used to perform enzymatic
ligation.
Free probe is removed by washing and retained fluorescent signal is measured.
The
color of the dye retained identifies which probe (A vs. G vs. C vs. T) was
attached
during the ligation reaction. RNase H2 is then used to cleave the probe,
removing the
"N" bases and universal bases but leaving the known base attached to the
acceptor
nucleic acid. In this manner the identity of the corresponding base within the
template
is determined, the acceptor nucleic acid has been extended by one base, and an
accessible 3'-hydroxyl is once again available for ligation, permitting
cycling of the
process.
[0541] The following oligonucleotides shown below in Table 65, were made as
a
representative synthetic system to demonstrate ligation and subsequent
cleavage of
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CA 02949315 2016-11-23
RNA-containing fluorescent ligation probes using the methods of the present
invention.
The ligation probes here have a fixed 9 base sequence (without any "N" bases
or
universal base modifications). The designation "CLP" indicates "cleavable
ligation
probe". The designation "ANA" indicates an "acceptor nucleic acid" which
provides
the 3'-hydroxyl acceptor site for a ligation reaction. "Targ-A" is a target
nucleic acid,
which directs a ligation reaction involving the complementary "T" ligation
probe
("CLP-T-Cy3"). "Targ-T" is a target nucleic acid, which directs a ligation
reaction
involving the complementary "A" ligation probe ("CLP-A-FAM").
Table 65:
CLP-C-TR 5' -pCaGCTGAAG-TR SEQ ID
No. 272
CLP-G-Cy5 5' -pGaGCTGAAG-Cy5 SEQ ID
No. 273
CLP-A-FAM 5' -pAaGCTGAAG-FAM SEQ ID
No. 274
SEQ ID
CLP-T-Cy3 5' -pTaGCTGAAG-Cy3
No. 275
ANA 5' -CCCTGTTTGCTGTTTTTCCTTCTC SEQ ID
No. 276
Targ-A 5' -AGTGTTTGCTCTTCAGCTAGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 277
Targ-T 5' -AGTGTTTGCTCTTCAGCTTGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 278
DNA bases are shown in uppercase. RNA bases are shown in lowercase. "p" is
5'-phosphate. TR is the fluorescent dye Texas Red. Cy5 is the fluorescent dye
Cyanine-5. Cy3 is the fluorescent dye Cyanine-3. FAM is the fluorescent dye
6-carboxyfluoreseein. The position of base variation between Targ-A and Targ-T
is
underlined, which is complementary to the 5'-basc of the corresponding
ligation probe.
[0542] Figure 36 shows the predicted results for a ligation-cleavage
reaction cycle
using the synthetic oligonucleotide sequences shown above. "Targ-A" (SEQ ID
No.
277) will direct hybridization and ligation of the "CLP-T-Cy3" probe (SEQ ID
No. 275)
while "Targ-T" (SEQ ID No. 278) will direct hybridization and ligation of the
"CLP-A-FAM" probe (SEQ ID No. 274). Assuming that the reactions have high
specificity, the remaining two ligation probes do not have a matching target
in this
experiment and so should not participate in the ligation reaction. Following
ligation,
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CA 02949315 2016-11-23
the newly formed fusion of the "ANA" + "CLP" product will become a substrate
for
RNase H2. Cleavage by RNase H2 will result in release of the 3'-end of the
ligation
probe (including the RNA base and the fluorescent reporter dye), leaving the
"ANA"
molecule longer by one base.
[0543] The "T" target nucleic acid (SEQ ID No. 278) or the "A" target
nucleic acid
(SEQ ID No. 277) and the "ANA" acceptor nucleic acid (SEQ ID No. 276) were
mixed
at 1.75 jiM and all 4 ligation probes (SEQ ID Nos. 272-75) were added to a
concentration of 3.5 p.M (each) in T4 DNA Ligase buffer (50 mM Tris-HCI pH
7.5, 10
mM MgC12, 10 mM dithiothreitol, 1 mM ATP) in a volume of 80 L, heated to 70 C
for
3 minutes and cooled slowly to 25 C. Ligation reactions were incubated at 37
C for 5
minutes with or without 140 units of T4 DNA Ligase. The reactions were stopped
by
heating at 65 C for 10 minutes. Reaction volumes were then adjusted to 200 pi
with
the addition of RNase H2 buffer [Tris-HC1 pH 8.0 (final concentration 10 mM),
NaC1
(final concentration 50 mM), MgCl2 (final concentration 4 mM)] and 20 units of
RNase
H2 was added to each tube. Reaction mixtures were incubated at 60 C for 30
minutes,
TM
followed by desalting over a Sephadex G25 column, and the samples were
lyophilized.
Samples were rehydrated in 70 jiL of water and 10 itL aliquots were analyzed
on a 20%
acrylamide, 7M urea, denaturing gel, followed by visualization using GelStar
stain
(#50535 GelStar Nucleic Acid Gel Stain, Lonza). The remainder of the reactions
was
saved at -20 C for future testing, including mass spectrometry or other
methods as
needed.
[05441 The gel image is shown in Figure 37. Lanes 1 and 5 show the
component
oligonucleotides in the absence of enzymes to visualize migration relative to
size
markers (lane M). Lanes 2 and 3 are duplicate reactions where Targ-A (SEQ ID
No.
277) was incubated with the 4 cleavage ligation probes (SEQ ID Nos. 272-75) in
the
presence of T4 DNA Ligase. An upward size shift of the acceptor nucleic acid
(ANA,
SEQ ID No. 276) is clearly seen which represents ligation with CLP-T-Cy3 (SEQ
ID
No. 275) and is identified as the ligation product. Specific ligation with the
correct
CLP-T-Cy3 probe and not the other 3 probes (mismatched bases) occurred, which
was
verified by visual inspection of the color of the dye (this cannot be
appreciated in the
black and white image shown in Figure 37) and was further verified by mass
spectrometry. Similarly, lanes 7 and 8 are duplicate reactions where Targ-T
(SEQ ID
169
CA 02949315 2016-11-23
No. 278) was incubated with the 4 cleavage ligation probes (SEQ ID Nos. 272-
75) in
the presence of T4 DNA Ligase. An upward size shift of the acceptor nucleic
acid
(ANA, SEQ ID No. 276) is clearly seen which represents ligation with CLP-A-FAM
(SEQ ID No. 274) and is identified as the ligation product. Specific ligation
with the
correct CLP-A-FAM probe and not the other 3 probes (mismatched bases)
occurred,
which was verified again by visual inspection of the color of the dye and
confirmed by
mass spectrometry analysis. Finally, lanes 4 and 8 demonstrate that these
ligation
products are reduced in size when treated with RNase H2, indicating that
cleavage
occurred. Note that the resulting bands show slightly reduced mobility
compared with
the original ANA band, indicating that this new species is longer than the
starting
material. Mass spectrometry confirmed that actual mass of the reaction
products in
lanes 4 and 8 were consistent with the predicted 1-base elongation of the
starting ANA
nucleic acid, that the correct base was inserted, and that the new "ANA+1"
species had
a 3'-hydroxyl. The new ANA+1 species is now prepared for a second cycle of
ligation/cleavage.
10545l This example has therefore demonstrated that short RNA-containing
short
probes can be specifically ligated to an acceptor nucleic acid in the presence
of a
complementary target nucleic acid. Ligation is sensitive to the identify of
the template
base matching the 5'-terminal base of the ligation probe and specific ligation
of the
correct complementary probe can be detected from within a heterogeneous mix of
different probe sequences. Finally, RNase H2 can cleave the ligation probe at
the
5'-side of the RNA base, releasing the bulk of the probe, resulting in an
acceptor
nucleic acid molecule which has been extended by one base in length. The
extended
acceptor nucleic acid contains a 3'-hydroxyl and can be used in repeated
cycles of
ligation/cleavage.
EXAMPLE 30 Use of universal bases in RNase H2-cleavable ligation probes
105461 In Example 29 above it was proposed that universal bases, such as
5'-nitroindole or inosinc, could be used in cleavable ligation probes. The
present
example demonstrates use of the universal base 5-nitroindole in a model system
where
the probe sequence is fixed (does not contain random N-bases). The
oligonucleotides,
shown below in Table 66, were synthesized based upon the synthetic
probe/template
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CA 02949315 2016-11-23
system in Example 29. Cleavage ligation probes were designed as 8mers with a
5'-phosphate, an "A" base at the 5'-end (to direct ligation to the "T"
target), a single
ribonucleotide, and 2 or 3 additional fixed DNA bases. Three or four 5-
nitroindole
bases were positioned towards the 3'-end. A FAM fluorescent dye was attached
at the
3'-end. The same acceptor nucleic acid (ANA) and T-target nucleic acid were
employed as in Example 29. A reaction scheme showing alignment of
oligonucleotide
components for this example is shown in Figure 38.
Table 66:
CLP-A-FAM SEQ ID
5f-pAaGCTXXX-FAM
-3x5NI No. 279
CLP-A-FAM SEQ ID
5f-pAaGCXXXX-FAM
-4x5NI No. 280
ANA 5f-CCCTGTTTGCTGTTTTTCCTTCTC SEQ ID
No. 276
Targ-T 5f-
AGTGTTTGCTCTTCAGCTTGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 278
DNA bases are shown in uppercase. RNA bases are shown in lowercase. "p" is
5'-phosphate. "X" is the universal base 5-nitroindole. FAM is the fluorescent
dye
6-carboxyfluorescein. The position of base hybridization with the 5 '-end of
the ligation
probe is underlined on the target.
[0547] The "T" target nucleic acid (SEQ ID No. 278) and the "ANA" acceptor
nucleic acid (SEQ ID No. 276) were mixed at 2 uM with the 3X or 4X 5 '-
nitroindole
containing CLPs (cleavable ligation probes, SEQ ID Nos. 279-80) in T4 DNA
Ligase
buffer (50 mM Tris-HC1 pH 7.5, 10 mM MgC12, 10 mM dithiothreitol, 1 mM ATP).
The reactions were heated at 70 C for 5 minutes and cooled slowly to 25 C. T4
DNA
Ligase (New England Biolabs) was added at a range of 7.5-120 units and the
ligation
reactions were incubated at 25 or 37 C for 5 minutes. The reactions were
terminated by
the addition of EDTA to a final concentration of 50 mM. Final reaction volumes
were
50 L. An equal volume of 90% formamide, 1X TBE loading buffer was added to
each
sample, which were then heat denatured at 70 C for 3 minutes and cooled on
ice.
Samples were separated on a denaturing 7M urea, 20% polyacrylamide gel. Gels
were
stained using GelStarTm stain and visualized with UV excitation. The gel image
is
shown in Figure 39.
171
CA 02949315 2016-11-23
[0548] The 8mer cleavable ligation probe with three 5-nitroindole universal
bases
(SEQ ID No. 279) worked well and showed near 100% ligation efficiency using
the
higher enzyme amounts (60-120 units T4 DNA Ligase). In contrast, the 8mer
cleavable
ligation probe with four 5-nitroindole universal bases (SEQ ID No. 280) did
not ligate
to the acceptor nucleic acid using any amount of enzyme. The same results were
seen at
25 C and at 37 C suggesting that this difference in reactivity does not relate
to
difference in Tm of the two probes. It is more likely that the differential
reactivity
relates to substrate preferences for the T4 DNA Ligase enzyme. This Example
demonstrates that three 5-nitroindole bases can be positioned at the 3'-end of
an 8mer
ligation probe and retain good function. This same experiment was repeated
using
9mer ligation probes. In this case, a probe having "six DNA + three 5-
nitroindole
bases" and a probe having "five DNA + four 5-nitroindole bases" were both
substrates
for T4 DNA Ligase but a probe with "four DNA bases + five 5-nitroindolc bases"
did
not (data not shown), consistent with the idea that T4 DNA Ligase requires 5
fixed
DNA bases towards the 5 '-end of the ligation probe to function well and that
5'-nitroindole bases can be introduced after this requirement is met. The
precise
optimal probe design can vary with different ligase enzymes.
[0549] These findings are significant as it permits synthesis of lower
complexity
pools of ligation probes.
EXAMPLE 31 Use of random bases and universal bases in RNase H2-cleavable
ligation probes
[0550] Examples 29 and 30 demonstrated use of RNase H2-cleavable ligation
probes where some or all of the probe sequence was a perfect match to the
target. In
sequencing a nucleic acid of unknown sequence, it is necessary to use probes
that
contain primarily random bases so that probe hybridization can occur for any
sequence
encountered. The present Example demonstrates use of 8mer cleavable ligation
probes
having a random base (Nmer) domain, a universal base (5-nitroindole) domain
and only
a single fixed DNA base at the 5 '-end. The following oligonucleotides shown
in Table
67 were employed:
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CA 02949315 2016-11-23
Table 67:
CLP-A-FAM SEQ ID
5'-pAnNNNXXX-FAM
4N+3x5N1 No. 281
CLP-T-Cy3 SEQ ID
5'-pTnNNNXXX-Cy3
4N+3x5NI No. 282
CLP-G-Cy5 SEQ ID
5'-pGnNNNXXX-Cy5
4N+3x5NI No. 283
ANA 5'-CCCTGTTTGCTGTTTTTCCTTCTC SEQ ID
No. 276
Targ-T 5'-
AGTGTTTGCTCTTCAGCTTGAGAAGGAAAAACAGCAAACAGGG SEQ ID
No. 278
DNA bases are shown in uppercase. RNA bases arc shown in lowercase. "p" is
5'-phosphate. "N" represents a random mix of the DNA bases A, G, C, and T. "n"
represents a random mix of the RNA bases A, C, G, and U. "X" is the universal
base
5-nitroindole. FAM is the fluorescent dye 6-carboxyfluorescein. Cy5 is the
fluorescent
dye Cyanine-5. Cy3 is the fluorescent dye Cyanine-3. The position of base
hybridization with the 5'-end of the ligation probe is underlined on the
target.
105511 The "T" target nucleic acid (SEQ ID No. 278) and the "ANA" acceptor
nucleic acid (SEQ ID No. 276) were mixed together at a final concentration of
0.4 111\4
each and the three cleavable ligation probes (SEQ ID Nos. 281-83) were
individually
added at a final concentration of 50 !AM (125-fold excess over the target and
acceptor)
in T4 DNA Ligase buffer in a final reaction volume of 50 4. Reactions were
heated to
70 C for 5 minutes and cooled slowly to 25 C. T4 DNA Ligase was added (400 U)
and
the ligation reactions were incubated at 37 C for 30 minutes. The reactions
were
stopped by heating at 65 C for 10 minutes followed by desalting over a
Sephadex G25
column, after which the samples were lyophilized and rehydrated in 10 tL of
water
mixed with 10 !..J.L of 90% formamide, lx TBE loading buffer. Samples were
heat
denatured at 70 C for 3 minutes and cooled on ice. Reaction products were
separated
on a 20% acrylamide 7M urea denaturing gel, followed by visualization using
GelStar
stain with UV transillumination (50535 GelStar Nucleic Acid Gel Stain, Lonza).
Results are shown in Figure 40.
[0552] The target nucleic acid contained a "T" base at the site
complementary to
the point of ligation. This template correctly directed ligation of the "A-
FAM" ligation
probe (SEQ ID No. 281) but not the mismatch "T-Cy3" (SEQ ID No. 282) or "G-
Cy5"
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CA 02949315 2016-11-23
(SEQ ID No. 283) ligation probes. Ligation specificity was directed by a
single fixed
DNA base at the 5'-end of the ligation probes which otherwise comprised random
"N"
bases or universal 5-nitroindole bases. The ligation probes were added to the
ligation
reactions at 125-fold molar excess over the target and the acceptor nucleic
acids. The
ligation probes contain a 4-base "N" domain, so the complexity of the nucleic
acid
mixture was 44 (256). Thus the reaction theoretically contained sufficient
perfect
matched probe to ligate with only about 50% of the input acceptor nucleic
acid. It is
evident from the relative fluorescent images in Figure 40 that approximate
half of the
acceptor was present in the longer ligation product species and half was
unreacted,
indicating that the reaction proceeded as expected. If mismatched sequences
ligated to
the acceptor with any appreciable efficiency, then the 125-fold excess of
ligation probe
would most likely have reacted with >50% of the acceptor nucleic acid
molecules,
which was not observed. Thus ligation reactions using cleavable ligation
probes of this
design were both efficient and specific.
EXAMPLE 32 Use of RNase H2-cleavable probes in an oligonucleotide ligation
assay (OLA)
105531 Use of cleavable ligation probes in DNA sequencing represents just
one
potential format/application for this general class of assay. The sequencing
application
is unique in that the target nucleic acid is of unknown sequence. More
typically,
oligonucleotide ligation assays are employed to determine the presence or
absence of a
known nucleic acid sequence within a sample nucleic acid of interest. For
example, an
OLA can be employed to detect the presence of a nucleic acid sequence specific
for
pathogenic organisms in the background of human DNA. Another example would be
to determine the presence or absence of a known, defined polymorphism at a
specific
target nucleic acid locus (e.g., an allelic discrimination assay or SNP
assay). In all of
these applications, one ligation probe is positioned so that the 3'-most or 5'-
most base
aligns with the SNP site and a second perfect-match nucleic acid is positioned
adjacent
so that if the probe sequence is a match for the SNP base, then a ligation
event can occur.
If the probe sequence is a mismatch for the SNP base, then ligation is
inhibited. The
ligation event results in formation of a detectable species.
105541 An allelic discrimination (SNP) assay is shown in this Example to
demonstrate utility of the novel RNase H2 cleavable ligation oligonucleotide
probes of
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CA 02949315 2016-11-23
the present invention. Sequence designs shown herein place the SNP site
towards the
3'-end of the acceptor ligation probe.
[0555] A traditional OLA employs three synthetic oligonucleotides to
discriminate
between two alleles (Figure 41A). If the SNP site comprises a "C" allele and
an "A"
allele, then two acceptor oligonucleotides are required, one bearing a "G"
base (match
for the "C" allele) and one bearing a "T" base (match for the "A" allele). The
acceptor
oligonucleotides have a free 3'-hydroxyl group. A third oligonucleotide (a
donor
nucleic acid) is employed that hybridizes to the target so as to place its 5'-
end adjacent
to the 3'-end of the ligation probe. The acceptor nucleic acid will have a 5'-
phosphate;
generally the 3'-end of the donor oligonucleotide is blocked so that it cannot
participate
in a ligation reaction. In this way, perfect match hybridization of both a
acceptor and
the donor probes on the target will position the two oligonucleotides in a
head-to-tail
fashion that enables ligation between the 3'-hydroxyl of the acceptor with the
5'-phosphate of the donor (Figure 41B). In contrast, a mismatch at the SNP
site
disrupts this structure and inhibits ligation. In the traditional OLA, the
identity of the
SNP base is interrogated once at the time of hybridization/ligation and
specificity is
entirely dependent upon the ability of the DNA Ligase to perform ligation on
the
perfect matched but not the mismatched species. Typically the three
oligonucleotides
(two ligation probes and the acceptor) have a similar Tm so that they can
function
together with the target nucleic acid under identical conditions.
105561 The new RNase H2 OLA of the present invention employs four synthetic
nucleic acids to discriminate between two alleles (Figure 42A). If the SNP
site
comprises a "C" allele and an "A" allele, then two cleavable acceptor ligation
probes
are required in this embodiment, one bearing a "G" base (match for the "C"
allele) and
one bearing a "T" base (match for the -A" allele). The cleavable acceptor
ligation
probes have a single RNA base positioned towards the 3'-end of the molecule
that is
aligned to be complementary or not (match vs. mismatch) with the base at the
target
SNP site. Additional DNA bases are positioned 3'- to the RNA base (preferably
four
DNA bases, all being complementary to the target) and a blocking group is
placed at the
3'-end to prevent ligation. The general design and function of the cleavable
ligation
probe is similar to the cleavable primers demonstrated in Example 13 in a qPCR
format
SNP discrimination assay. The cleavable ligation probes can also be designed
using
175
CA 02949315 2016-11-23
various chemically modified bases and abasic residues as outlined in the above
Examples to improve SNP discrimination at the RNase H2 cleavage site (see
Examples
22, 23, 25, and 28). Preferably the cleavable ligation probes will be designed
to have a
Tm in the range of 60-70 C (in RNase H2 cleavage buffer) to permit
hybridization of
the cleavable probe with target in the optimal temperature range for the
enzyme.
[0557] Unlike the traditional OLA format, the donor oligonucleotides in the
RNase
H2 OLA format are also SNP interrogation probes. Thus two donor probes are
required,
one bearing a "G" base (match for the "C" allele) and one bearing a "T" base
(match for
the "A" allele). Both donoror probes have a phosphate at the 5'-end to enable
ligation
and optionally are blocked at the 3'-end (Figure 42A). The two donor ligation
probes
can have a lower Tm than the RNase H2 cleavable ligation probes so that
hybridization
of the cleavable ligation probes and the donor ligation probes with the target
nucleic
acid can be differentially regulated by control of reaction temperature. These
donor
probes in the assay format do not interact with RNase H2.
[0558] To perform an RNase H2 OLA, all four OLA probes are mixed in the
presence of the target nucleic acid in a buffer compatible with RNase H2
activity (see
above examples). Preferably this will be done around 60-70 C. The RNase H2
cleavable acceptor oligonuleeotide is complementary to and will hybridize to
the target
nucleic acid under these conditions. If the RNA base of the acceptor probe and
the base
at the target SNP site match, then RNase H2 cleavage can occur (Figure 42B).
It is
preferred that the donor ligation probe (the non-cleavable probe) has a lower
Tm than
the cleavable probe. The first stage of the reaction (hybridization of the
acceptor
oligonucicotide and cleavage by RNase H2) can then be carried out at a
temperature
that is sufficiently above the Tm of the non-cleavable donor probes that they
do not
hybridize to target. Cleavage of the acceptor probe by RNase H2 removes the
RNA
base and uncovers the SNP site, making it available to hybridize with the non-
cleavable
ligation probe (the donor oligonueleotide).
[0559] Once the RNase H2 cleavage phase of the OLA is complete, reaction
temperature is lowered to permit hybridization of the non-cleavable ligation
probe to
the target. In the presence of DNA Ligase, the 5'-end of the non-cleavable
probe will
ligate to the 3'-end of the adjacent cleaved probe (Figure 42B), if the base
at the 5'-end
of the donor probe pairs with the base at the SNP site. Thus the RNase H2 OLA
assay
176
CA 02949315 2016-11-23
interrogates the identity of the base at the SNP site twice, once during RNase
H2
mediated cleavage of the acceptor oligonucleotide probe and again at the
ligation
reaction (Figure 42C). Double interrogation of the identity of the SNP base by
two
different enzymatic events provides for greater specificity than can be
achieved using a
traditional OLA.
EXAMPLE 33 SNP discrimination using RNase H2-cleavable probes in an OLA
105601 A variety of
methods exist that enable detection of OLA products. In the
present Example, fluorescence detection is performed in a bead capture assay
format to
perform an RNase H2 OLA allelic discrimination assay as outlined in Example
32.
Sequences were designed that were compatible for use with the Luminex xMAP
fluorescent microbead system with detection on a Luminex L100 detection system
(Luminex, Austin, TX).
105611 The "OLA-C-
antitag" and "OLA-T-antitag" sequences (SEQ ID Nos. 284-5)
were made with a 5 '-amino modifier to permit conjugation to carboxylate xMAP
fluorescent beads using carbodiimide coupling chemistry. The "OLA-T-Tag" and
"OLA-C-Tag" sequences (SEQ ID Nos. 288-9) which serve as donor
oligonucleotides
in the ligation reaction have a 12-base sequence towards the 5'-end which is
complementary to the target sequence and positions the SNP site (C/T base) at
the
5'-end. Tm for these 12-base domains is estimated to be 50-53 C (in 10 mM Mg
containing buffer). Both sequences have a 5'-phosphate to permit ligation. The
3'-end
of these sequences is a "tag" sequence which is complementary to the "antitag"
sequence and permits capture to antitag bearing beads by hybridization. The
"OLA-C"
and "OLA-T" probes (SEQ ID Nos. 286-7) serve as the acceptor fragment and are
complementary to the target and position the single ribonucleotide base (rC or
rU) at
the SNP site. Tm for the cleavable ligation probes is estimated to be ¨75 C
(in 10 mM
Mg containing
buffer). Both of the oligonucleotide probes have a biotin at the 5'-end
which will enable binding of a reporter dye, Streptavidin-phycoerythrin, for
detection
by the Luminex L100 system. Synthetic 98mer oligonucleotide targets
corresponding
to the "C" allele (G base in the target, SEQ ID No. 290) and "T" allele (A
base in the
target, SEQ ID No. 291) were employed in this Example. The sequences
corresponding to SEQ ID Nos. 284-291 are shown below in Table 68. Alignment
and
177
CA 02949315 2016-11-23
interaction of the different probe, target, tag, and antitag sequences during
the various
step in this assay are shown in Figure 43..
Table 68:
OLA-C SEQ ID
5' aminoC12-GATTTGTATTGATTGAGATTAAAG
antitag No. 284
OLA-T SEQ ID
5' aminoC12-GATTGTAAGATTTGATAAAGTGTA
antitag No. 285
rs4939827 SEQ ID
5' Biotin-CACCATGCTCACAGCCTCATCCAAAAGAGGAAAcAGGA-x
OLA C N286
rs4939827 SEQ ID
5' Biotin-CACCATGCTCACAGCCTCATCCAAAAGAGGAAAuAGGA-x
OLA T No. 287
rs4939827
SEQ ID
OLA C 5' pCAGGACCCCAGACTTTAATCTCAATCAATACAAATC-x
No. 288
Tag
rs4939827
Q
OLA T 5' pTAGGACCCCAGATACACTTTATCAAATCTTACAATC-x SE ID
No. 289
Tag
5'CCCAGCATTGTCTGTGTTTCCTGAGGAGTCTGAGGGAGCTCTGGGGTC SEQ ID
Targ-C
CTGTTTCCTCTTTTGGATGAGGCTGTGAGCATGGTGGATTAGAGACAGCC No. 290
T 5'
CCCAGCATTGTCTGTGTTTCCTGAGGAGTCTGAGGGAGCTCTGGGGTC SEQ ID
arg-T
CTATTTCCTCTTTTGGATGAGGCTGTGAGCATGGTGGATTAGAGACAGCC No. 291
DNA bases are uppercase. RNA bases are lowercase. Biotin is a Biotin-TEG
group. X
represents a C3 spacer. For the OLA C/T Tag oligonucleotides, the portion of
the
sequence which is the "tag" and binds the "antitag" sequence is underline. The
site of
the SNP with the target sequence under interrogation is underlined and in
bold.
[0562] Coupling of antitag oligos to xMAP microspheres. Anti-tag
oligonucleotides containing a 5' amino group were coupled to 1.25x107 xMAP
Multi-Analyte COOH Microspheres (L100-C127-01 and L100-C138-01, Luminex,
Austin, TX) using 3 mg/mL N-(3-Dimethylaminopropy1)-N'-ethylcarbodiimide
hydrochloride (03449-1G, Sigma Aldritch),in 0.1 M MES, pH 4.5 buffer (M-8250
Sigma-Aldritch) at room temperature for 90 minutes in the dark (modified
manufacturer's protocol). After coupling, the microspheres were washed once
with
0.02% Tween20, and then once with 0.1% SDS. Microspheres were re-suspended in
200 4. of TE pH 7.5. The concentration of microspheres was determined by
counting
with a hemocytometer under a light microscope (Nikon TMS, Freyer Company,
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CA 02949315 2016-11-23
Carpentersvillc, IL). Successful coupling was determined by hybridizing 25-250
fmolcs of complementary oligonucleotides containing a 5' biotin modification
and
detecting the hybrids with 2 1..tgimL streptavidin R-phycoerythrin conjugate
(S866 1
mg/mL, Invitrogen, Carlsbad, CA). Mean fluorescence intensity had to increase
in a
concentration dependent manner. No cross hybridization was observed between
the
two anti-tag sequences.
[0563] OLA Assay. RNase H2 digestion mixtures (10 jtL) were prepared
containing rs4939827 OLA C and rs4939827 OLA T oligos (SEQ ID Nos. 286-7) at a
final concentration of 250 nM, and either C, T or C/T mix template
oligonucleotides
(SEQ ID Nos. 290-91) at 125 nM in a 20 mM Tris-HCI (pH 7.6 at 25 C), 25 mM
KAc,
mM MgAc, 10 mM DTT, 1 mM NAD, and 0.1% Triton X-100 buffer (Taq DNA
Ligase buffer, New England Biolabs, Ipswitch, MA). Samples were incubated for
30
minutes at 65 C with or without 5 mU of Pyrococcus akvssi RNase H2. For each
RNase H2 digestion reaction, the volume was increased to 25 jiL by adding 2.5
pmoles
of rs4939827 OLA 12C Tag and 2.5 pmoles rs4939827 OLA 12T Tag oligonucleotides
(SEQ ID Nos. 288-9) (100 nM final concentration for each oligo), with or
without 40 U
of Taq DNA Ligase (New England Biolabs, Ipswitch, MA), maintaining a final
buffer
composition of 20 mM Tris-HC1 (pH 7.6 at 25 C), 25 mM KAc, 10 mM MgAc, 10 mM
DTT, 1 mM NAD, and 0.1% Triton X-100. The ligation reactions were incubated at
45 C for 30 minutes.
[0564] Capture of ligation product on fluorescent beads and detection of
signal. 10 nL of each ligation mixture was combined with 15 jit of H20, and 25
pi of
the xMAP bead mixture (Bead sets 127 and 138) at a density of 100 beads of
each
type/AL. The samples were heated to 70 C for 90 seconds followed by 50 C for
30
minutes. The samples were transferred to a Milliporem Multiscreen filtration
plate
(MABVN1250, Millipore, Bedford, MA), and washed two times with 100 jit of 50 C
0.2 M NaCI, 0.1 M Tris pH 8.0, 0.08% Triton X-100 buffer. Microspheres were
incubated at 50 C for 15 minutes with 75 jiL of a 2 ng/mL solution of
streptavidin-R
phycoerythrin (S866 1 mg/mL, Invitrogcn, Carlsbad, CA). Mean fluorescence was
measured on a Luminex L100 detection system (Luminex, Austin, TX).
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CA 02949315 2016-11-23
105651 Results are shown in Figure 44. Fluorescent beads bearing the "C"
allele
antitag sequences showed positive fluorescent signal only when the reaction
was run in
the present of the "C" allele target or the "C/T" mix. Fluorescent beads
bearing the "T"
allele antitag sequences showed positive fluorescent signal only when the
reaction was
run in the present of the "T" allele target or the "C/T" mix. Signal was
dependent on
use of RNase H2 and was not observed in the absence of target DNA. Thus the
RNase
H2 cleavable oligonucleotide ligation assay of the invention was demonstrated
to be
effective at distinguishing the presence of a C/T SNP present in a target DNA
in a
highly specific fashion.
Additional Acknowledgements
105671 The use of the terms "a" and "an" and "the" and similar referents in
the
context of describing the invention (especially in the context of the
following claims)
are to be construed to cover both the singular and the plural, unless
otherwise indicated
herein or clearly contradicted by context. The terms "comprising," "having,"
"including," and "containing" are to be construed as open-ended terms (i.e.,
meaning
"including, but not limited to,") unless otherwise noted. Recitation of ranges
of values
herein arc merely intended to serve as a shorthand method of referring
individually to
each separate value falling within the range, unless otherwise indicated
herein, and each
separate value is incorporated into the specification as if it were
individually recited
herein. All methods described herein can be performed in any suitable order
unless
otherwise indicated herein or otherwise clearly contradicted by context. The
use of any
and all examples, or exemplary language (e.g., "such as") provided herein, is
intended
merely to better illuminate the invention and does not pose a limitation on
the scope of
the invention unless otherwise claimed. No language in the specification
should be
construed as indicating any non-claimed element as essential to the practice
of the
invention.
180
CA 02949315 2016-11-23
[0568] Preferred embodiments of this invention are described herein,
including the
best mode known to the inventors for carrying out the invention. Variations of
those
preferred embodiments may become apparent to those of ordinary skill in the
art upon
reading the foregoing description. The inventors expect skilled artisans to
employ such
variations as appropriate, and the inventors intend for the invention to be
practiced
otherwise than as specifically described herein.
181
