Note: Descriptions are shown in the official language in which they were submitted.
1111PRO..
WO 93/15102 PC1/US93/00486
DESCRIPTION
Method For Use Of Branched Nucleic Acid Probes
Field of the Invention
This invention concerns nucleic acid probes,
generally designed for use in hybridization assays.
Background of the invention
Nucleic acid is formed from nucleotide bases, e.g.,
uracil (U) cytidine (C), adenine (A), thymine (T) and
guanine (G), formed as a single-stranded linear molecule.
Such a linear molecule can form a hybrid,complex, or
double-stranded molecule (also called a duplex), with
another linear molecule by forming specific base pairs,
e.g., between A and T, A and U, or G and C. Such paired
molecules are called complementary molecules.
Nucleic acid hybridization is a method in which two
single-stranded complementary molecules form a double-
stranded molecule.=This method is commonly used to detect
the presence of one single-stranded molecule in a sample
(the target nucleic acid) by use of a labelled probe
formed of a complementary single-stranded molecule. Fqn
example, Muran 'et al., WO 87/04165, published July 16,
20. 1987, describe a probe having a single-stranded region
complementary to the nucleic acid to be detected, and a
double-stranded region having a non-radioactive label.
The general uses and design of nucleic acid probes are
well known in the art, see e.g., Mifflin 35 Clin. Chem.
1819, 1989, and Matthews and Kricka, 169 Anal. Biochem. 1,
1988.
Summary of the Invention
This invention features novel nucleic acid probes and
methods for their use. It is based upon the use of one or
more probes which can form a detectable structure only in
the presence of a target nucleic acid.. In general, at
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least two portions of a nucleic acid molecule (which may be
on different molecules) must hybridize with the target
nucleic acid to create this structure. Thus, the probe
ensures that little or no false positive results are
observed. Such probes can be used to detect nucleic acids,
and also can be used as therapeutic agents. They allow
novel detection methods to be practiced, independent of the
structure of the target nucleic acid. In addition,
amplification of a label signal can be readily achieved,
such that the probes allow extremely sensitive levels of
detection of target nucleic acid.
Thus, in a first aspect, the invention features a
nucleic acid hybridization probe having at least one nucleic
acid strand which has at least two separate target-specific
regions that hydridize to a target nucleic acid sequence,
and at least two distinct arm regions that do not hybridize
with the target nucleic acid but possess complementary
regions that are capable of hybridizing with one another.
These regions are designed such that, under appropriate
hybridization conditions, the complementary arm regions will
not hybridize to one another in the absence of the target
nucleic acid; but, in the presence of the target nucleic
acid the target-specific regions of the probe will anneal to
the target nucleic acid, and the complementary arm regions
will anneal to one another, thereby forming a branched
nucleic acid structure. The formed structure is a
detectable branched nucleic acid structure which does not
depend on labels that interact to produce a detectable
signal and which does not depend on detection by a method
dependent on primer extension of either of the arm regions.
In a related aspect, the invention features a
method for use of such a probe to detect the presence or
amount of a target nucleic acid in a sample. In this method
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one or more nucleic acid molecules are provided which
together include at least two separate regions which
hybridize with the target nucleic acid under a predetermined
environmental condition. The molecules also include at
least two arm regions, which (under the predetermined
environmental condition) do not hybridize with the target
nucleic acid, or with each other in the absence of the
target nucleic acid. In the presence of the target nucleic
acid, however, the arm regions do hybridize with each other.
The method further includes contacting the nucleic acid
molecules with a sample under the predetermined
environmental conditions to allow hybridization of the arm
regions if the target nucleic acid is present. Any
hybridization of the arm regions is then detected as an
indication of the presence or amount of the target nucleic
acid. The detecting is not dependent upon labels that
interact to produce a detectable signal. The method does
not involve detecting molecules resulting from primer
extension of either of the arm regions.
In preferred embodiments, the detecting step
includes contacting the arm regions with a resolvase (by
which term is meant an enzyme able to resolve a junction by
junction-specific cleavage, e.g., a 4-way junction or a 3-
way junction) and detecting cleavage of the one or more
nucleic acid molecules by the resolvase, performing a DNA
footprint analysis of the one or more nucleic acid
molecules, observing the mobility of the one or more nucleic
acid molecules within a gel matrix, detecting the binding of
an intercalator to the one or more nucleic acid molecules,
contacting the one or more nucleic acid molecules with S1
nuclease and detecting any cleavage of the one more nucleic
acid molecules by the nuclease, contacting the one or more
nucleic acid molecules with a restriction endonuclease and
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detecting cleavage of the one or more nucleic acid molecules
by the endonuclease, or determining the thermal stability of
the one or more nucleic acid molecules.
In other preferred embodiments, the one or more
nucleic acid molecules include an intercalating molecule or
nucleic acid binding molecule, e.g., an acridinium ester, or
other molecule which is susceptible to chemical modification
by an acid or a base, or other modification, when that
molecule forms part of a single-stranded nucleic acid
molecule or a double-stranded nucleic acid molecule, and is
not susceptible to such chemical cleavage when the molecule
is present in the other of the single-stranded nucleic acid
molecule or double-stranded nucleic acid molecule; and the
detecting step includes contacting the one or more nucleic
acid molecules with the chemical and
WO 93/15102 PCF/US93/00486
4
determining the amount of chemical modification of the
molecule; at least one of the one or more nucleic acid
molecules has a target region having between eight and 30
(or 8-100) complementary bases in a region of 50 (or 1000)
contiguous bases of the target nucleic acid; the arm
regions form a duplex in the absence of target nucleic acid with a melting
temperature (commonly referred to as
Tm, which is measured as described below) 4 C lower than
the hybridization temperature in the predetermined
environmental condition, most preferably 7 C or even 10 C
lower, and in the presence of target nucleic acid form a
duplex having a Tm at least 4 C (or 7 C, or even 10 C)
greater than the hybridization temperature,in the pre-
determined environmental condition.
In yet other preferred embodiments, one nucleic acid
molecule is provided and the nucleic acid molecule has a
loop region connecting the at least two arm regions; the
one or more nucleic acid molecules consist of two nucleic
acid molecules each having a target region and an arm
region; the one or more nucleic acid molecules consist of
three nucleic acid molecules each having at least one arm
region, and at least two of the nucleic acid molecules
having a separate target region, wherein the three nuclei.a
acid molecules hybridize with the target nucleic acid to
form at least two separate hybridized or duplex arm
regions; the one.or more'nucleic acid molecules consist of
four nucleic acid molecules each having,at least one arm
region, and at least two of the nucleic acid molecules
have separate target regions, wherein the four nucleic
acid molecules and the target nucleic acid hybridize to
form at least three separate duplexes between the arm
regions.
In still other preferred embodiments, the target
regions hybridize with the target nucleic acid, and the
.35 'arm regions hybridize together to form an arm, such that
a junction is formed at the base of the arm between the
two separate target regions. The one or more nucleic acid
WO 93/15102 PCI'/US93/00486
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molecules or the target nucleic acid may include nucleic
acid adjacent the junction which does not form a duplex
with the arm regions or the target regions or the target
nucleic acid, and loops out from the junction. Alterna-
5 tively, the target regions include along their length, or
at the ends distant from the arm regions, nucleic acid
which does not form a duplex with the target nucleic acid
and therefore either loops from a duplex formed between
the target nucleic acid and the target region, or extends
as a single-stranded region from the end of the target
region. In yet another alternative, the arm regions
include nucleic acid which does not form a duplex with the
other arm region and forms a loop eXtending from the arm
region or extends as a single-stranded molecule from the
end of the arm region distant from the target region. In
one example, the target regions hybridize with.the target
nucleic acid, and the arm regions hybridize together to
form an arm, and a junction is formed at the base of the
arm=between'the two separate target regions. One or both
arm regions further has a single-stranded region at the
end furthest from the target region which fails to
hybridize to the other arm region, and thus is available
for duplex formation with another nucleic acid molecule ta
form a second akrm. In this example, the one or more
nucleic acid molecules may include a portion able to form
a duplex with the single-stranded regions to form a second
or third arm and a second junction between the arms.
In related preferred embodiments, the at least two
arm regions form an arm when hybridized with the target
nucleic acid, and this arm includes a biologically or
chemically active site, e. ., a restriction endonuclease
site, a duplex region and a single-stranded region,
wherein the duplex region acts as a primer for a DNA
polymerase, a promoter for an RNA polymerase, e.g., which
can be transcribed to form a plurality of'RNA transcripts,
a DNA-RNA duplex susceptible to cleavage.by RNAseH, and a
WO 93/15102 PC T/US93/00486
6
chemical active to cleave adjacent duplex nucleic acid,
e=a=, Fe=EDTA.
In addition, an arm region or target region may
include a single-stranded region which is able to loop
over a duplex region to form triple-stranded nucleic acid.
Such triple-stranded structures are well known in the art and can be readily
designed as part of the present inven-
tion. Such a single-stranded nucleic acid may also be
provided separate from the rest of the nucleic acid mole-
cules, if desired, and can be labelled by standard method-
ology. Alternatively, it may form part of an arm or even
target region. Detection of triple stranded nucl,eic acid
is readily performed by methods known in the art.
The method may also include the step of contacting
the one or more nucleic acid molecules and the target
nucleic acid with other nucleic acid molecules able to
hybridize with the arm regions, or single-stranded regions
extending from these arm regions, to form one or more
duplex regions or arms, and these other nucleic acid
molecules are then able to further hybridize among them-
selves to form a plurality of arms or arm regions.
Thus, the above probes can be incubated under suit-
able hybridization conditions with a sample suspected oF+
containing the target nucleic acid, ,and detection of
hybridization of the complementary arm regions used as an
indication of the presence of target nucleic acid. Gen-
erally, the probe 'is formed from two or more separate
nucleic acid molecules, e=a=, strands of lengths between
20 and 50 bases; and the presence of hybridization is
monitored by detection of an acridinium ester within the
strands.
In another related aspect, the invention features a
method for creating a biologically or chemically signifi-
cant site which may be distant from the target nucleic
acid yet is formed only in the presence of the target
nucleic acid. This site is a segment of nucleic acid
which is relatively inert in relation to a specific chem-
WO 93/15102 PCT/US93/00486
7
ical or biochemical activity when it is in the single-
stranded form, but becomes active in relation to specific
chemical or biochemical activity when it is hybridized
with its complementary strand, or vice versa. Examples of
chemical or biochemical activity include, but are not lim-
ited to, a site susceptible to double-strand specific
chemical cleavage (e.g., using an Iron-EDTA complex, or
providing phenanthroline to duplex nucleic acid to make
the duplex susceptible to copper cleavage), restriction
endonuclease digestion, RNAse H digestion (one strand of
the duplex is RNA and one strand is DNA), or a site which
can be acted upon by a T7 RNA polymerase, or a DNA poly-
merase activity, e.g., allowing primer extension activity
(e=o=, using Taq DNA polymerase). Other examples include
sites which are digested when in the form of single-
stranded nucleic acid (with S1 nuclease) but is protected
from such digestion when in a double-stranded form. The
nucleic acid hybridization probe having this site is
designed as described above, except that it includes such
a site, or such a site is created when the probe is hybri-
dized with a complementary arm region. The probe is thus
incubated with target nucleic acid to form a branched
nucleic acid structure, as well as the desired site.-+
Further chemistry or biochemistry is then performed as
desired. This can be used, e.g., as a mode of detection,
a mode of amplification, or a mode of target-mediated
cleavage of nucleic'acid.
In yet another related aspect, the invention features
a method for mediating a biological activity within a tar-
get cell by hyridizing a probe, similar to those discussed
above, to a specific target site. As described'above, a
probe is incubated with target nucleic acid to form a
branched nucleic acid structure, including a duplex region
formed between complementary arm regions. Examples of
~35 target nucleic acid in this method include essential
sequences within the genome of a pathogenic organism,
essential mRNA sequences produced by a pathogenic organ-
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= . -
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WO 93/15102 PCl'/US93/00486
8
ism, and essential sequences within cancer cells. (By
essential is meant that the viability of the organism or
cell is reduced by at least 30%, as measured by techniques
well known in the art, when such a sequence is deleted.)
The duplex region formed between complementary arm regions
of the probe is designed as a specific recognition site
for one of a variety of therapeutic approaches, including
a sequence-specific antibody recognition site (a specifi-
cally bound antibody will mediate therapy through any of
a variety of well known mechanisms, e.g., a cell toxin
linked to the antibody will kill the cell), and a sequence
specific protein/enzyme binding (therapy is mediated by
enzymatic cleavage, blocking of transcription, blocking of
translation, and the like). Furthermore, the junction
between nucleic acid strands can serve as the recognition
site for such.antibodies or proteins.
Other features and advantages of the invention will
be apparent from the following description of the pre-
ferred embodiments thereof, and from the claims.
Deacritition of the Preferred Embodiments
The drawings will first briefly be described.
_ . ,=~
Dra~
FIGS. lA-iF, 4A-4B, 6A-6H, 9A-9F, and 11A-11F are
gsneral diagrammatic representation of probes of this
invention (the short lines between probes in each of the
figures represent hydrogen bonding between the probes, and
are not representative of actual number of bases or hydro-
gen bonds);
FIGS. 2A-2E and 3A-3B, 5A-5G, 7 and 8, 10, and 12A-
12C are specific examples of the probe shown respectively
in FIGS. 1A, 4A, 6A, 9F, and ilE;
FIGS. 13 and 14 are specific examples of mismatched
probes;
FIGS. 15A-15F are diagrammatic representations of
various detection systems useful in the invention;
WO 93/15102 PCT/US93/00486
4 o 1 2 8,
9
FIG. 16 is a specific example of probes detectable by
restriction endonuclease treatment;
FIG. 17 is an autoradiogram of a test with the probe
shown in FIG. 16;
FIG. 18 is a specific example of a probe detectable
by enzymatic amplification;
FIGS. 19A and 19B, and 20A-20C are diagrammatic
representations of target-independent probe amplification
systems;
FIG. 21 is a diagrammatic representation of three
probes producing a 4-way junction; and
FIGS. 22A-221 are diagrammatic representations of
various embodiments of the probes shown in FIG. 21.
Probes
Referring to FIGS. 1A and 1B, there is shown one
example of a probe of this invention demonstrating the
general structure of each probe of this invention. The
probe shown is formed from two separate strands 10, 12
each of which possess a'target-specific region14, 16 able
to hybridize under hybridizing conditions to a target
nucleic acid 15. Each strand also possesses an arm region
19, 20 which hybridize together to form an arm 22 gener='
ally only in the -presence of the target nucleic acid.
When an arm is formed so also is a junction 21 between the
arm and hybridized target regions. Arm regions and target
specific regions can be designed so that hybridization of
the target specific regions to target nucleic acid is
observed under hybridization conditions only when both
strands are present in the hybridization mixture, and both
are hybridized to target nucleic acid and to each other at
their arm regions.
Thus, as generally discussed above, nucleic acid
hybridization probes of this invention form branched
nucleic acid structures (i.e., structures having at least
one arm extending from a target nucleic acid and not
directly hybridized with the target nucleic acid) upon
WO 93/15102 PtT/US93/00486
interaction with and hybridization to a target nucleic
acid. The probes are designed so that formation of this
branched structure is target-dependent. Therefore, detec-
tion of branch formation is a measure of the presence and
5 the amount (if detection is quantitative) of target ,
nucleic acid in a sample. More than one branch can be
formed, but only one branch must be formed in a target-
dependent manner. The probe can be a single nucleic acid
strand or can be multiple strands. Furthermore, the
10 strand (or strands) can be DNA, modified DNA, RNA, modi-
fied RNA, or various combinations thereof. By "modified
DNA or RNA" is meant any nucleic acid structure differing
from naturally occurring DNA or RNA molecules, e.g., DNA
or RNA, containing modified bases (e.g., methyl cytosine,
or inosine), or modified internucleotide linkages (e.g.,
phosphorothioate, or methyl phosphonate). The probe must
have at least two separate regions that hybridize specif i-
cally with the target nucleic acid, and at least two arm
regions (i.e., regions that do not hybridize with the
target) that are complementary to one another and form a
stable duplex only in the presence of target. Since these
complementary arm regions hybridize only in the presence
of target nucleic acid, detection of this duplex region is
the preferred method to assay for the presence of' the
25, target nucleic acid.
There are numerous methods for detecting hybridiza-
tion of branched nucleic acid probes with target. Exam-
ples of detection of branched nucleic acids include use of
junction specific cleavage with a resolvase enzyme (e. .,
where cleavage of a labelled probe strand is detected),
DNAse footprinting, gel retardation assays, and binding of
a labeled, junction specific intercalating compound (where
the target is captured and the presence of label
detected). While purified 4-way junction cleaving resol-
.35 vases are known in the art, 3-way junction cleaving
enzymes and other junction specific enzymes can be readily
purified by art known methods, for example, by purifying
_ . . _.._. _ _. . ___._..... ....,. .... . .._._. . .._ ...
WO 93/15102 PCT/US93/00486
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2 8
11
enzymes biochemically which have an activity which cleaves
a desired 3-way junction. Examples of detection of the
duplex formed between complementary arm regions include
DNAse footprinting, Si nuclease digestion (an arm region
is labelled; if the arms form a duplex, they will not be
digested with nuclease Si; if the arms do not form a
duplex, they will be digested with nuclease Si), restric-
tion endonuclease. cleavage of a site created by duplex
formation between complementary arm regions, capture of
target nucleic acid followed by analysis of the thermal
stabilities of the associated duplex(es) (using a labelled
probe strand(s); the complete branched nucleic acid struc-
ture will have a higher thermal stability than any partial
formation; if no formation has occurred, a different sig-
nal will be associated with the captured target nucleic
acid compared to when a branch is formed). Also the
juxtapositioning of two synergistic labels on opposite
probe arms can be detected. More generally, it will be
readily recognized by those skilled in the art that any
method that detects the formation of branched DNA, or the
formation of a complementary arm duplex, can be utilized
in this method to indicate the presence of target.
- The preferred method of detection is the use of the
Hybridization Protection Assay (HPA) described by Arnold
25. et al., 35 Clin. Chem. 1588, 1989. This method utilizes
a chemiluminescent acridinium ester (AE) label covalently
attached to a synthetic oligonucleotide probe. The assay
format is completely homogeneous (i.e., requires no physi-
cal separation steps) and is based on chemical hydrolysis
of the ester bond of the AE molecule, cleavage of which
renders the AE permanently non-chemiluminescent. Condi-
tions have been developed where the hydrolysis of this
ester bond is rapid for unhybridized AE-labeled probe
(AE-probe) but slow for hybridized AE-probe. Therefore,
after hybridization of the AE-probe with its complementary
nucleic acid (under conditions'that do not promote ester hydrolysis), the
reaction conditions are.adjusted so that
WO 93/15102 PCT/US93/00486
=,,
12
the chemiluminescence associated with unhybridized
AE-probe is rapidly reduced to low levels, while the
chemiluminescence associated with hybridized AE-probe is
minimally affected. Following this differential hydroly-
sis process, any remaining chemiluminescence is a direct
measure of the amount of duplex formed. This assay method
is used in the present invention by attaching one or more
AE labels to one or more of the nucleic acid strands of
the probe and assaying for duplex formation using the
differential hydrolysis procedure described above.
Two Comoonent Probes
One configuration of branched nucleic acid probe used
for the detection of target nucleic acids is shown sche-
matically in FIGS. 1A and iB. In this configuration the
probe consists of two separate nucleic acid strands; these
strands are preferably synthetic oligonucleotides. Each
strand possesses a probe region which hybridizes with the
target nucleic acid and an arm region. The two arm
regions are complementary to one another (by which is
meant not that they are necessarily perfectly complemen-
tary, since they may possess portions that are not comple-
mdntary to each other at all, but-that under hybridizingl
conditions a stable duplex is formed).- These arm'regions
are designed such that.the melting temperature, or Tm, of
the associated duplex in the absence of target is less
than the operating temperature of the assay, preferably
4 C less (more preferably 7 C or 10 C) than the operating
temperature, so that little or no hybridization of the arm
regions occurs in the absence of target nucleic acid. The
Tm is defined as the temperature at which 50% of a given
nucleic acid duplex has melted (i.e., has become single-
stranded). The Tm is dependent on environmental condi-
tions such as the cation concentration of the solution.
The desired Tm is typically achieved by manipulation of
the length and nucleotide base composition of the comple-
mentary regions. Other methods can also be utilized to
1rt4'1 L'- .15 q F .. L~'x - '~ . . . .
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.
WO 93/15102 PCT/US93/00486
2~2 o
13
adjust duplex Tm, including but not limited to incorpora-
tion of mismatches, replacement of some or all of the
guanosine residues with inosine, and modifying the phos-
phate backbone, e.g., with phosphorothioate internucleo-
tide linkages. When utilizing the HPA format at 60 C, the
preferred length of.exactly complementary, unmodified arm
regions is approximately 8 to 20 contiguous bases (depen-
dent on base composition and sequence). Other environ-
mental conditions would potentially lead to a different
size range; this is easily determined empirically.
Upon contacting the probe with a solution containing
the target nucleic acid, the probe regions of the two
strands will hybridize to their.r.espective target regions,
which are typically adjacent to one another, as shown in
FIG. 1 (they do not have to be immediately adjacent, see
in.tra). When this occurs, the arm regions of the two
probe strands are constrained to be in close proximity to
one another, thus increasing the stability of the associ-
ated duplex. The arm regions are designed such that the
Tm of the duplex formed in the presence of target is
approximately equal to or above the operating temperature
of the assay, preferably 4 C above (more preferably 7 C or
10, C) the operating temperature such that the arms will+
form a duplex. As indicated above, when utilizing the HPA
tormat at 60 C, the preferred length of the arms is
approximately S. to 20 contiguous complementary bases
(dependent on base composition and sequence).
The probe regions of the two separate strands can be
designed in a variety of manners. For example, these
regions can be designed similarly to the arm regions in
that the Tm of either region alone (i.e., one probe strand
plus the target strand) is below the operating tempera-
ture, but is above the operatingtemperature when both
probe strands and the target strand are present and the
.35 arm regions are hybridized. They can also be designed
such that the Tm's of the probe regions are both above the
operating temperature, or they can be designed such.that
WO 93/15102 PCT/US93/00486
cl
P - 14
one Tm is above and one Tm is below the operating tempera-
ture. Whatever design is chosen, the requirement that the
arm regions form a stable duplex only in the presence of
target must be met. The probe regions are preferably
between 8 and 50 nucleotides in length, more preferably
between 8 and 30 nucleotides in length. These regions can
be longer, but most applications do not require this addi-
tional length, and synthesis of these longer oligomers is
more costly and time consuming than the shorter oligomers.
One or both probe sequences is chosen to react with
the desired target nucleic acid(s) and preferably to not
react with any undesired target nucleic acid(s) (i.e.,
cross-react). If one probe region hybridizes with an
undesired target but the other probe region does not, the
assay will still function properly since both probe seg-
ments have to hybridize in order for the arm regions to
hybridize.
When utilizing the HPA format, the AE label is
typically placed in the arni region .of the probe. In the
absence of target, this region will be-single-stranded and
the AE will be susceptible to rapid ester hydrolysis. In
the presence of target, this -region will be double-
stranded and the AE will be protected from ester hydroly--"'
sis. An AE label can be placed in one strand, the other
strand, or both. strands (thereby leading to an increase in
signal and assay sensitivity). Multiple labels can be
placed in each strand, further improving assay sensitiv-
ity. The AE label can also be placed in one or the other
(or both) of the probe regions. Multiple labels could
also be used,in this configuration. Furthermore, AE could
be utilized in any combination of the placements just
described.
To detect a target nucleic acid in a sample using the
branched nucleic acid probe described above in the HPA
35= format, the following general procedure'is used: 1) add
the branched nucleic acid probe to the sample, 2) incu-
bate to allow annealing of the appropriate regions to
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WO 93/15102 PCT/US93/00486
2 J,"~ J'n
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occur, 3) add a reagent to effect differential hydrolysis
of the AE label(s), and 4) detect any remaining chemilumi-
nescence as an indication of the presence or amount of any
target nucleic acid present. The annealing conditions can
5 be varied depending on the exact application, the design
of the probe, the nature of the target nucleic acid and
the composition of the sample in which the target is con-
tained. The conditions must be chosen, however, to ful-
fill the Tm requirements stated above. For the HPA for-
10 mat, the incubation temperature is preferably between 5
and 70 C, more preferably between 30 and 65 C; the pH of
the annealing step is preferably between 4.5 and 8.5, and
most preferably between 4.5 and 5.5 (greater stability of
the AE during the annealing-step is achieved in the lower
15 pH range; however, methods exist to stabilize the AE in
the higher pH range, as described by Hammond et al., 6 J.
Hiolum, and Chemilum. 35, 1991). The concentration of the
monovalent salt counterion is preferably between 50 and
1000mM, more preferably between 200 and 800mM; it is pre-
ferred to include detergent.in the annealing step (this
prevents the AE from aggregating and from sticking to the
walls of the tube, and the pipet tips), preferably at con-
centrations between 0.1 and 10% (w/v), and more preferably
between 1 and 10%. One skilled in the art will recognize
that alternate annealing conditions that lead to the
desired hybridization characteristics may also be used in
this invention.
Although a specific example is provided below, this
is not limiting in the invention. Those of ordinary skill
in the art will recognize that many variations are possi-
ble, examples of which are provided in FIGS. iC-iF, where
various loops (labelled L) are shown in an arm, target
nucleic acid or at a junction, and where single-stranded
nucleic acid (labeled S) may also be present (e.g., for
35, later duplex function with more probes as described
below).
WO 93/15102 ' PCr/US93/00486
16
ExamDle 1
A specific example of the general configuration shown
in FIG. 1 is shown in FIG. 2A. The target strand is a
synthetic DNA oligomer 43 bases in length with a sequence =
corresponding to a region of the 23S ribosomal RNA (rRNA)
subunit of Mycobacterium tuberculosis. The 2 strands of
the branched nucleic acid probe form a 3-way junction with
the target strand as shown in FIG. 2A. A single AE is
covalently tethered to strand 1 between a T and an A
residue as shown.
Oligonucleotides were synthesized using standard
phosphoramidite solid-phase chemistry (Caruthers,et al.,
154 In Methods Enzvmol. 287, 1987) on a Biosearch Model
8750 or ABI 380A DNA Synthesizer and purified using stan-
dard polyacrylamide gel electrophoresis. Strand 1 was
AE-labeled as pre-viously described by Arnold and Nelson,
PCT Publication No. WO 89/02896, published April 6, 1989.
The following annealing reactions were carried out
for 1 hour at 60 C in 30 1 of 0.1M lithium succinate buf-
fer, pH 5.2, 2mM EDTA, 2mM EGTA, and 10% (w/v) lithium
lauryl sulfate: Hybrid - 0.5 pmol of target strand, 0.05
pmol of probe strand 1 (the AE-labeled strand), and 2.5
pmol of probe strand 2. Control 1 - 0.05 pmol of probeq
strand 1, and 2e5 pmol of probe strand 2. Control 2 -
0.05 pmol of probe strand 1. Reagent Blank - No target or
probe.
To demonstrate'that the arm regions were hybridizing
only in the presence of target, the half-life of AE
hydrolysis for each of the reactions above were-measured
using the protocol described previously (Arnold et al., 35
Clin Chem. 1588, 1989). The following results were
obtained:
Hybrid: 11.5 minutes Control 1: 0.54 minutes
Control 2: 0.51 minutes
These data reveal that the AE is not-protected from
ester hydrolysis in the absence of target (compare Control
CA 02128530 2003-10-29
73091-27
17
1 with Control 2) , but is protected from hydrolysis in the
presence of target (Hybrid), thus demonstrating the hybri-
dization of the arm regions only in the presence oftarget
nucleic acid. 5 This system was further evaluated by measuring the
Tm's of the various configurations represented schemati-
cally in FIG. 2B. The Tm's were measured according to the
following protocol:
1. After annealing as described above, samples to be
analyzed were diluted in 0.1M lithium succinate buffer, pH
5.2, 2mM EDTA, 2mM EGTA, 10% (w/v) lithium lauryl sulfate
to approximately 100,000 relative light units (RLU; this
is the unit of chemiluminescence used in these protocols;
see Arnold et al., 35 Clin Chem. 1588, 1989) per 100 l.
2. Separate 100 l aliquots were pipetted into 12 X
75mm assay tubes. Each sample to be analyzed was incu-
bated for 7 minutes at each of the following temperatures
- 40, 43, 46, 49, 52, 55, 58, and 62 C for oligomers with
Tm's of 56 C and below; and 50, 53, 56, 59, 62, 65, 68,
and 71 C for oligomers with Tm's above 56 C. -After incu-
bation, each tube was placed in an ice bath.
3. After all incubations were complete, all tubes
were removed from the ice bath and to each 300A1 of 0.15M
sodium tetraborate buffer, pH 7.6, 5$ (v/v) Triton MX-100
added, and vortex mixed. All tubes were incubated at 40 C
for 20 minutes for oligomers with Tm's of 56 C and below,
and at 50 C for 15 minutes for oligomers with Tm's above
56 C. The tubes were placed on ice for 30 seconds, and
then at room,temperature (20-25 C).
4. Chemiluminescence was measured in a luminometer
(LEADEAOI, Gen-Probe, CA) by the automatic injection of
200 1 of 0.1% H202, 1N NaOH, followed by measurement of
signal for 5 seconds. The tubes were blotted with a moist
tissue or paper towel before measuring the chemi-
luminescence.
5. The Control value was subtracted from each cor-
responding Hybrid value, and the resulting data plotted as
WO 93/15102 PCT/US93/00486
'18
chemiluminescence versus temperature. The Tm was deter-
mined graphically as the temperature at which 50% of the
maximum chemiluminescence is lost.
The temperature range and the exact temperature
intervals used can vary with the exact characteristics of
the AE-oligomer being characterized.
The following Tm's were obtained:
Structure 1 (FIG. 2B) Tm = 62 C
Structure 2 (FIG. 2C) Tm = 50 C
These data demonstrate that the duplex associated
with the arm regions has a Tm well below 60 C (the oper-
ating temperature used above in the hydrolysis rate
determination) in the absence of target, and a Tm above
60 C in the presence of target.
The system was further characterized by measuring
Tm's of the various structures depicted in FIG. 2B-2E in
8mM lithium succinate buffer, pH 5.5, 100mM NaCl, 10mM
MgClz, and lOmM Tris-HCl (this reagent replaces the
diluting reagent in step 1 of the above protocol). The
following Tm's were obtained:
Structure 1 (FIG 2B) Tm = 59 C
Structure 2 (FIG 2C) Tm = 47 C
Structure 3 (FIG 2D) Tm = 47 C ,.,
Structure 4. (FIG 2E) Tm = 45 C
For this particular design, not only is the Tm of the
arm region much lower in the absence of target than in the
presence of target, but the duplex regions formed between
the target and the probe regions are less stable in the
absence of the complete arm duplex (see FIG. 2) than in
the presence of the complete arm duplex. This demon-
strates that the branched nucleic acids of this invention
can be used to manipulate the Tm of a nucleic acid duplex.
Other possible configurations for acridinium ester
label locations, or use of a restriction enzyme (BstIII)
,35 for duplex detection are shown in FIGS. 2B-2E.
WO 93/15102 PC'lf'/US93/00486
;~, ~ n .=>. ,~,~ ~,
19
Examiple 2
Another example of the general structure shown in
FIG. 1 is shown in FIGS. 3A and 3B. In this design ribo-
somal RNA (rRNA) from Neisseria gonorrhoeae is the target
nucleic acid. All oligomers were synthesized and
AE-labeled as described in Example 1 above. Two basic
designs were evaluated, with junctions at slightly differ-
ent locations within a given region of the rRNA (99/135
FIG. 3B and 132/146 FIG. 3A designs). A variety of
linker-arm placements were evaluated, as indicated below.
Furthermore, the exactly complementary target nucleic acid
gonorrhoeae) as well as a potentially cross-reacting
target nucleic acid with 2 mismatches (N. meninaitidis)
were evaluated.
Hybridization characteristics of the different
regions were evaluated using differential hydrolysis and
Tm analyses as described in Example 1 with the following
specific conditions:
Taroet nucteic acid (RNA) Oliao 99 Oliao 135 li 14
Samole Iygg Amount Label Amount Label Amount Label Amount
1 N. gon. 0.6 pmol AE 0.1 pmol none 2 pmol none 2 pmol
2 N. gon. 0.6 pmol none 2 pmol AE11) 0.1 pmol none 2 pmol
3 N. gon. 0.6 pmol AE 0.1 pmol AE(1) 0.1 pmol none 2 pmoj,
4 N. gon. 0.6 pmol none 2 pmol AE(2) 0.1 pmol none 2 pmol
5 N. men. 0.6 pmol AE 0.1 pmol none ' 2 pmol none 2 pmol
6 N. men. 0.6 pmol none 2 pmol AE(1) 0.1 pmol none 2 pmol
7 N. men. 0.6 pmol AE 0.1 pmol AE(1) 0.1 pmol none 2 pmol
8 N. gon. 0.6 pmol none 2 pmol AE(2) 0.1 pmol none 2 pmol
0li 132 01i 146 li 0
9 N. gon. 0.6 pmol AE 0.1 pmol none 2 pmol none 2 pmol
10 N. gon. 0.6 pmol none 2 pmol AE 0.1 pmol none 2 pmol
11 N. gon. 0.6 pmol AE 0.1 pmol AE 0.1 pmol none 2 pmol
12 N. men. 0.6 pmol AE 0.1 pmol none 2 pmol none 2 pmol
13 N. men. 0.6 pmol none 2 pmol AE(1) 0.1 pmol none 2 pmol
14 N. men. 0.6 pmol AE 0.1 pmol AE(1) 0.1 pmol none 2 pmol
WO 93/15102 PCI'/US93/00486
Hybridizations with N. gonorrhoeae were performed at
60 C; hybridizations with N. meningitidis were performed
at 45 or 50 C; controls contained all the components
except target; linear oligonucleotides 100 and 146 are
5"helper" probes that open the secondary structure of the
rRNA, and are not related to the branched nucleic acid
probes of this invention. The following results were
obtained:
Hydrolysis Half-life (mi.n)
10 Sample Target Tm C Hybrid Control D.H. Ratio
1 N.gon 68.2 14.3 0.48' 29.8
2 N.gon 70.0 6.5 0.51 12.7
3 N.gon 68.0 8.2 0.50 16.4
4 N.gon 68.5 18.8 0.47 40.0
15 9 N.gon 70.0 12.8 0.49 26.1
10 N.gon 72.0 8.0 0.43 18.6
11 N.gon 67.5 8.6 0.43 20.0
5 N.men 59.0
6 N.men 59.0
20 _ 7 N.men 54.5 .8 N.men 56.0
12 N.men 58.5
13 N.men 61.0
14 N.men 57.0
DH ratio is the ratio of half-life for hybrid and control.
Neisseria meningitidis DH ratios were near unity (data not
shown). Thus, no hybridization was observed.
The DH ratios for the N. gonorrhoeae target clearly
demonstrate that the arm regions form a stable duplex only
in the presence of an RNA nucleic acid target. These two
designs each have a strand with a long probe region and a
strand with a short probe region, thus demonstrating the
~a'~.. .,..'... ._. =..~.a1:~t r._.;4t~ sa.r_r!5.J.~%'= :_:. 2..r., e.:=. r
a.. -.' =e.a .., e ..5..:h.-., .J .,ra.lrrr., . ..,.vr.~.,...,.. .5e....,
....,.oa,. .,.,..,. ........... 4... .. .. ... -. .
CA 02128530 2003-10-29
73091-27
21
flexibility of this design parameter. Furthermore, the
data demonstrate that several different AE placements
yield similar results, including 2 AE's per probe (one per
each strand; samples 3 and 11). Some AE placements yield 5 higher DH ratios
than other placements, but all show pro-
tection only in the presence of target. The Tm data for
the N. gonorrhoeae target show that all structures tested
yield Tm's well above the operating temperature (60 C in
this case).
The data generated with the N.meningitidis target
demonstrate that the system can be tuned to not signifi-
cantly cross-react with a closely related target nucleic
acid, thus improving its utility as an assay. The differ-
ences in Tm for each structure between N. oonorrhoeae and
N. meningitidis range between 9.2 and 13.5 C.
The 99/135 system was also evaluated for its ability
to detect decreasing amounts of target nucleic acid (N.
cTonorrhoeae); cross-reaction with 10'1 /lg (63fmol) of N.
meningitidis was -also tested. The assay format was as
follows: Target and probe were annealed in 100 l of 0.1M
lithium succinate buffer, pH 5.2, 2mM EDTA,.2mM EGTA, and
10% (w/v) lithium lauryl sulfate for 60 min at 600C.
300 1 of 0.15M sodium borate buffer, pH 7.6, containing 5%
Tritori~X-100 detergent were added, vortexed and incubated
at 60 C for 10 minutes. Chemiluminescence was measured in
a luminometer (LEADER I, Gen-Probe, CA) by the automatic
injection of 200 1 of 0.4N HNO3, 0.1% H202, and then 200 1
of 1N NaOH, followed by measurement of signal for 5
seconds.
In this example, the following probe mixtures were
used: Probe mix 1- 0.1pmol of AE-labeled oligo 99,
0.5pmo1 of oligo 135, and 2 pmol of oligo 146; Probe mix
2 - 0.lpmol of AE-labeled oligo 135, 0.5pmol of oligo 99,
and 2 pmol of, oligo 146; Probe mix 3 - e.ipmol of
AE-labeled oligo 99, 0.lpmol of AE-labeled oligo 135, and
2 pmol of oligo 146. The amounts of target assayed and
the results are indicated below:
W~ . ... ,. .. . . . .... .., . . . . ... =t .... . . . . .. . . . ,. . , .
...
WO 93/15102 PCT/US93/00486
'
22
Probe Mix
(N.yon. ] (1Cg) 1 2 3
X 10"2 370,848 188,161 475,161
10"2 66,398 37,016 112,844
5 10"3 9,242 4,730 14,249
[N.men. 1(ua)
10'I 2,358 5,363 2,362
These data demonstrate that the designs shown here
are useful in detecting small amounts of nucleic acid in
a full assay format. Furthermore, label amplification is
demonstrated in probe mix 3, which contains 2 AE-labeled
strands instead of 1(as in probe mixes 1 and 2). Cross-
reaction with the very closely related N. meningitidis RNA
was minimal for probe mixes 1 and 3; the slight cross-
reaction seen in probe mix 2 is approximately 100-fold
lower than the positive signal, and was reduced to minimal
levels by slightly increasing the operating temperature of
the assay.
One Component Probes
Another configuration of branched nucleic acid probe
used for the detection of target nucleic acids is showA-+
schematically in-FIGS. 4A and 4B. In this configuration
the probe consists of a single nucleic acid strand. The
desired characteristics are the same as those described
for the design shown in FIG. 1, but now.the 2 strands of
the probe are connected and therefore become 1 strand. A
segment 24 connecting the two halves of the probe can be
nucleotide or non-nucleotide in nature, and should be of
a length that yields the desired probe characteristics
described above. When utilizing-the HPA format at 60 C,
the preferred length of the complementary, unmodified arms 25,' 27 in this
conf iguration is somewhat shorter than
described for the design shown in FIG. 1 due to the intra-
molecular nature of the interaction. Therefore, the pre-
ferred arm length is approximately 4 to 16 bases. As
......,.... ,,, . .......... ..r_ . .. , .
WO 93/15102 ,,, PCT/US93/00486
2~.2 8 ~~
23
mentioned above, other environmental conditions would
potentially lead to a different size range; this is easily
determined experimentally by standard procedures.
Example 3
An example of the general structure shown in FIG. 4
is shown in FIG. 5A. The target strand is a synthetic DNA
oligomer 43 bases in length with a sequence corresponding
to a region of the 23S rRNA subunit of Mycobacterium
tuberculosis. All oligomers were synthesized and
AE-labeled as described in Example 1 above. The branched
nucleic acid probe forms a 3-way junction with the target
strand as shown in FIG. 5A. A single AE is covalently
tethered to the probe between a T and an G residue as
shown. An AE was also placed in the-target strand at one
of the two locations, indicated in certain analyses as
described below.
Hybridization characteristics of the structures
depicted in FIGS. 5B-5G were evaluated using DH and Tm
analysis protocols described in Example 1; the amounts of
each component used in the respective annealing reactions
are listed in FIGS. 5B-5G. The following results were
obtained:
~rf
Hydrolysis Half-life (min)
sawle Hybrid Control D.H. Ratio
1 5.8 1.2 4.8
2 10.2 0.32 31.9
3 8.1 0.56 14.5
These data demonstrate that the duplex formed between
arm regions is more stable in the presence of target than
the absence of target, as revealed by the increased AE
stability. The relative difference in stability is large
enough to be used to clearly detect the presence of target
nucleic acid, but is not as great as that seen in either
Exampl=e'1 or 2. Comparing the control hydrolysis rates of
samples 1, 2 and 3, it is clear that the arms are inter-
WO 93/15102 pCT/LJS93/00486
.. =
Cr
(IN IY.IJ 24
acting slightly under the operating conditions of this
experiment. This interaction can be essentially elimi-
nated by optimizing the operating conditions (e.g., higher
operating temperature, inosine residues included in the
arm regions, or phosphorothioate linkages included in the
arm regions). This improves discrimination between the
target present and target absent cases.
Other configurations of branched nucleic acid probes
used for the detection of target nucleic acids are shown
schematically in FIGS. 6A-6H. In these configurations the
probe consists of three or more nucleic acid strands which
form one or more nucleic acid junctions with the target
nucleic acid. As above, such structures can be designed
within a single nucleic acid molecule. Virtually any
combination of junctions is possible as long as the arm
regions form a stable duplex only in the presence of tar-
get (the possibility of some of the arm regions forming a
stable duplex even in the absence of target will be dis-
cussed later). The guidelines for designing such a system
are essentially the same as those described above for the
configuration shown in FIG. 1. In the case of four way
junctions (or even higher order junctions), some of the
strands of the probe will contain only arm regions and nQ4
target specific.- regions. Since the sequence(s) of the
strand(s) that contains only arm regions is independent of
target sequence, this sequence(s) can be used in combina-
tion with a variety of specific probe strands for detec-
tion of a variety of target sites. This "universal detec-
tion" approach has several advantages, including the abil-
ity to use very few (or even only one) AE-labeled univer-
sal detection oligomer for the detection of all target
sequences (this greatly minimizes design and synthesis
time, therefore minimizing cost) and freedom to select the
sequence that yields optimal differential hydrolysis, com-
.35, pletely independent of target sequence.
As described above, AE can be placed in some or all
of the arm regions as well as some or all of the probe
WO 93/15102 PCT/trS93/00486
21~
regions. One advantage of this configuration is that
there are many sites for AE, thus greatly increasing the
label amplification potential of the assay. In one par-
ticular usage, the sequence around the AE site can be the
5 same for each arm region, leading to uniformity of AE
hydrolysis characteristics (AE hydrolysis rates are
sequence-dependent) and improving overall precision of the
assay. The lengths of the probe regions can be manipu-
lated to achieve certain assay performance goals. For
10 instance, cross-reaction with closely related targets can
be minimized by adjusting the lengths of the various probe
segments. As an example, in FIGS 6A, 6B, and 6C, probe
regions 30, 32 of probe strands 1 and 3 can be relatively
long and therefore form stable duplexes with target and
15 even closely related non-target nucleic acids that possess
homologous sequences in these regions. Probe region 34 of
probe strand 2 can be relatively short, and span a region,
that contains one or more mismatches 36 to non-target
nucleic acids. This probe segment will form a stable
20 duplex with perfectly matched target, but by virtue of its
relative shortness will not form a stable duplex with
mismatched targets.
- The above examples show three way junctions betweei!
an arm and two target regions. Four way junctions can be
2,5 created with three probes (although they may be formed as
a single molecule) as shown in FIGS. 6E and 6F with sepa-
rate arm regions (labelled A) and probe regions (P). Sim-
ilar examples are shown in FIGS. 6H and 6G.
Example 4
A specific example of the general configuration shown
-in FIG. 6A is shown in FIG. 7. The probes are labelled 99
strand, 135 strand and 146 strand. As in Example 2, ribo-
somal RNA (rRNA) from Neisseria gonorrhoeae is the target
nucleic acid. All oligomers were synthesized and
AE-labeled as described in Example 1 above. Two basic
designs were evaluated, both contairiing three probe
WO 93/15102 PCT/US93/00486
n..
26
strands that form two three-way nucleic acid junctions
with the target strand. The only difference between the
two designs is that one contains loop-out (L) bases at the
junctions as indicated in FIG. 7. (More loop bases can be
included in probes of this invention if desired; as can
non-nucleotide bases. In addition, probes may be formed
which create a non-fixed, or mobile, junction.)
A variety of linker-arm placements were evaluated, as
indicated below. Furthermore, the exactly complementary
target nucleic acid (N. gonorrhoeae) as well as a poten-
tially cross-reacting target nucleic acid with 2 mis-
matches (N. meninaitidis) were evaluated.
Hybridization characteristics of the different
regions were evaluated using differential hydrolysis and
Tm analyses as described in Example 1 with the following
specific conditions: Structures 1-18 represented schema-
tically in FIG. 8 were analyzed always using 0.6 pmol of
the target strand, 0.1 pmol of AE-labeled probe strands,
and 2 pmol of unlabeled probe.strands; in samples 10-12
the target was N. meningitidis; in all the other samples,
the target was gonorrhoeae; samples 1,2,4-6,10,11,13,
14,16 and 17 contained loop-out bases as shown in FIG. 7;
the remaining samples did.not contain loop-out bases. The,,#
resulting data are represented in tabular form in FIG. 8.
These datademonstrate that multiple.arm regions can
form'in the presence of target, and only in the presence
of target. Furthermore, multiple AE labels can be used in
a single design, thus providing label amplification.
Also, these structures can form with and without the loop-
out bases shown in FIG. 7.
Selected structures from those shown in FIG. 8 were
also evaluated for their ability to detect target nucleic
acid (N. gonorrhoeae); cross-reaction with N. meningitidis was also tested.
The general assay format was the same as
that used in Example 3, with the following probe amounts:
AE-labeled strands - 0.1 pmol; non-labeled probe strands
WO 93/15102 2 1 S :~ PC'T/US93/00486
~
... K, ~
27
- 2 pmol. The amounts of the target nucleic acids assayed
and results are indicated below:
Structure No target
(see Fig.8) 10-2 Mg N.gon. 10-2ggN.men. Control
1 29,820 817 726
4 28,564 1302 683
5 15,540 1892 1127
16 90,000 1309 1211
These data demonstrate that the designs shown here
are useful in detecting small amounts of nucleic acid in
a full assay format. Furthermore, significant label
amplification is demonstrated in structure 16, which
contains 3 AE-labeled strands instead of 1 (as in probe
structures 1, 4 and 5). Cross-reaction with the very
closely related 1. meningitidis RNA is minimal for struc-
ture 16; the slight cross-reaction seen in the other
structures could be reduced to minimal,levels by slightly
increasing the operating teinperature of the assay, which
was selected as the optimal for structure 16.
- Non-target Junction.Probes ,.
Another con.figuration of branched nucleic acid probe
used for the detection of target nucleic acidsis repre-
sented schematically in FIGS. 9A-9F. This configuration
is very similar to the one just described above in that
the probe consists of three or more nucleic acid strands,
e.g., shown as 40, 42, 44, which form two or more nucleic
acid junctions, but in this case one or more of the junc-
tions are formed with the target nucleic acid (such as 1
in FIG. 9A) and one or more of the junctions are not asso-
ciated with the target (such as junction 2 in FIG. 9A) but
are still.formed only in the.presence of target. Virtu-
ally any combination of junctions is possible as long as
the arm regions form a stable duplex only in the presence
of target (the possibility of some of the arm regions
WO 93/15102 PCT/US93/00486
<i
4pw y 28
forming a stable duplex even in the absence of target is
discussed infra). Several examples of structures of this
nature are represented schematically in FIGS. 9A-9F.
The guidelines for designing such a system are essen-
tially the same as those for the configurations described
above. The best combination of lengths and sequence of
each region of each strand are typically determined exper-
imentally by testing a variety of structures until the
optimal combination is achieved. The design variables and
the advantages of this configuration are essentially the
same as those described above for the configuration repre-
sented by FIG. 6.
Exarnple 5
A specific example of. the general configuration shown
in FIG. 9 is shown in FIG. 10. The target is a synthetic
DNA 60mer (T) with a sequence corresponding tQ the major
genetic translocation associated with chronic myelogenous
leukemia (see Example 6 below). The probe consists of
three strands, two of which form a 3-way junction with the
target, and one of which forms a 3-way junction with the
other two. This third strand contains only arm regions,
and as such is a potential universal detection oligomer a$-4
discussed above.* A single AE labeling site is detailed in
this example, but AE can be placed in other regions as
well.
To evaluate' performance of this system, DH and Tm
analyses were performed as described in Example 1 with the
following specific conditions: target strand = 0.5 pmol;
unlabeled probe strands = 2 pmol; AE-labeled probe strand
= 0.1 pmol; differential hydrolysis was performed at 50 C.
The results were as follows:
Half-life (min) Tm C
Hybrid Control Ratio Hybrid Control
45.7 0.97 47.1 59.8 46.2
ARM1S..... ... . . .... . .. .. t....... .. ,.. ..,.. . , .... .. .. . . .. .
. ,. . ... . . , :ti: .;1.., . ... .. . .
WO 93/15102 PCT/US93/00486
2t ~8 5 'j''
~%
29'
These data demonstrate that the two junctions of this
structure form only in the presence of target (at the
operating temperature) as evidenced by the lack of protec-
tion against AE hydrolysis of the AE-labeled strand. This
is a unique situation in which the hybridization of a
strand that does not even come into contact with the tar-
get strand is completely target dependent. This clearly
demonstrates the concept of the universal detection oligo-
mer discussed above. This also demonstrates the ability
to form multiple junctions in response to target.
Chimeric Probes
In another configuration, a branched nucleic acid
probe can be used to detect a chimeric nucleic acid tar-
get. A chimeric nucleic acid is a nucleic acid made up
two or more segments that each originate from different
sources or are otherwise unique (e.g., the chimeric DNA
only exists in a portion of a population of organisms) in
some way. An example of such a chimeric target is the
product of a genetic translocation as represented schema-
tically in FIG. 11A. In this case, a segment of one
chromosome is translocated onto another chromosome (and
vice-versa in a reciprocal translocation), creating,-a
chimeric DNA molecule (this may or may not result, in a
chimeric mRNA). This type of translocation can occur with
multiple breakpoints (with one or, both chromosomes),
resulting in multiple chimeric DNA's. FIG. 11B represents
a case in which 3 separate breakpoints on chromosome 2
reassociate with one breakpoint on chromosome 1.
Chimeric targets of this kind can be detected in a
number of ways, examples of which are=given in FIGS. 11C-
11F. Method 1 (FIG. 11C) is a sandwich assay that
requires a physical separation step. This method utilizes
a capture probe specific for chromosome 1 (this capture
probe is labeled with a specific capture agent, C, which
allows it to be specifically removed from solution; for
example, C may be biotin and the specific capture support
WO 93/ 15102 !'CI'/US93/00486
y~vr.1
may be avidin agarose), and 3 separate detection oligomers
(each labeled with a reporter group) specific for the
three different translocated regions of chromosome 2.
Method 2 (FIG. 11D) is a homogeneous assay that util-
5 izes an AE-labeled probe specific for each translocation
product (this is accomplished by designing the probes to
"bridge" the breakpoint junction).
Method 3 (FIG. 11E and 11F) utilizes the method of
this invention, in which a branched nucleic acid structure
10 is formed around the breakpoint junction (other structures
are possible, such as those shown in previous FIGS. in
this application). Strand a is universal for all translo-
cation products, and strands d, f and h are specific for
the corresponding translocation products. In this parti-
15 cular example, strand a is the only strand that is
AE-labeled. This system has several advantages, including
the following: it is homogeneous, not requiring the
physical separation steps required in Method 1; it has a
universal sequence for AE protection, leading to uniform
20 differential hydrolysis characteristics for each target;
it has only 1 AE-labeled strand, minimizing complexity and
cost and lowering backgrounds; it is less sensitive to
mismatches (which can occur iri the breakpoint junctiou..*
region) than Method 2 (see Example 7). The guidelines for
25 designing such a system are essentially the same as those
for the configuration described in FIG. 1 above.
Examgle 6
A specific example of the general configuration shown
in FIGS il is shown in FIGS. 12A-12C. The chimeric
30 targets are synthetic DNA oligomers (80mers) homologous to
3 different genetic translocations between a constant abl
region of chromosome 9 and various regions of chromosome
22. Two are the most common translocations associated
with chronic myelogenous leukemia (CML) and one is associ-
ated with acute lymphocytic leukemia (ALL). Each chimeric
target contains an identical ab1 region, whereas all
WO 93/15102 PCT/US93/00486
31
regions from chromosome 22 are different. An 80mer
corresponding to the normal abl gene (bridging the
breakpoint 40 bases on either side) was also synthesized.
A single AE-labeled strand specific for the abl
region was designed- as shown in FIGS. 12A-12C. Three
different strands were designed to contain a probe region
specific for one of the translocated chromosome 22 regions
as well as an arm region complementary to the arm region
of the universal detection oligomer.
Performance of these 3 designs were first evaluated
by measuring the differential hydrolysis rates and Tm's
for each using the protocol described in Example 1 (target
- 0.5 pmol; AE-labeled probe strand - 0.1 pmol; unlabeled
probe strand - 2 pmol). The results appear below:
Half-life (min. )
Structure Hybrid Control DH Ratio TM(OC)
1 18.3 0.636 28.8 71.5
2 18.4 0.640 28.8 ---
3 18.6 0.640 29.1 20 Next, the ability of the 3 probe mixes to detect the
appropriate target sequences' (and not react with tl).e#
inappropriate target sequences) was evaluated. The assay
format described in Example 4 was used (target - 20 fmol;
AE-labeled probe strand - 0.1 pmol; unlabeled probe strand
- 2 pmol). The results appear below,(average of quadru-
plicate reactions):'
WO 93/15102 PCT/US93/00486
~ '. L%
c
~N 3 2
Probe Probe Probe AE-labeled
Target mix 1 mix 2 mix 3 strand only
1 202,187 1,354 1,265 889
2 2,950 167,614 1,343 1,107
3 1,307 2,319 131,670 729
normal ab1 1,491 1,248 1,094 886
no target 624 614 506 451
The probe mixes detect only the correct chimeric
targets and do not cross-react significantly with either
of the other two chimeric targets or the normal abl
sequence, thus demonstrating the utility of this confi-
guration for the detection of. chimeric nucleic acid
targets, and the use of a single AE-labeled probe strand
for the detection of multiple targets.
Mismatch Probes
In another configuration, a branched nucleic acid
probe is used to detect a variety of targets with related
but non-identical sequences. Typically, DNA probe-based
assays are designed to detect a specific target and to not
cross-react with closely related targets. In fact great
ef-fort has been expended to develop assays that will b?,.q
sensitive to. as -little as one mismatch. However, there
are cases when an assay needs to be relatively insensitive
to mismatches. An example of such a case is the detection
of viruses such as HIV, which display significant genomic
variation.
Example 7
A specific example of the general configuration dis-
cussed above is shown in FIG. 13. The exact target strand
is a synthetic DNA oligomer, 78 bases in length with a
sequence corresponding to a region of the gag gene in
HIV-1. Other synthetic 78mers were also constructed to
contain a variety of mismatches as-shown in FIG. 13 {these
sequences correspond to various HIV strains that have been
WO 93/15102 PC'i'/US93/00486
/ @ ~ =~ h ~ ~~
33
isolated and sequenced). Two branched nucleic acid struc-
tures were evaluated, which have identical probe regions
but different arm regions (see FIG. 14). The probe
regions were designed to be very stable at the operating
temperature, thus increasing the overall stability of the
branched nucleic acid structure once formed with the tar-
get. The stability of each region, including the stabil-
ity added by the arm regions (once hybridized), buffers
against loss of stability in other regions caused by mis-
matches, thus decreasing the sensitivity of the overall
structure to mismatches. All oligomers were synthesized
and AE-labeled as-described in Example.1 above.
Differential hydrolysis ratios and Tm's of these
designs were determined using the general protocols given
in Example 1 above with the following specific conditions:
target strand - 0.5 pmol; AE-labeled strand(s) - 0.1 pmol;
unlabeled strand - 2 pmol; hybridizations and correspond-
ing DH ratio analyses were performed at three different
temperaturet (60, 55 or 500C; see below). The results are
as follows:
_ ,.~
WO 93/15102 PCT'/US93/00486
c~ ;=,
34
Half-lifelminl
Probe AE- tr nd Taraet NOM' Temat Hybrid Control DH Ratio 96FIvb
1 1 1 0 60 4.3 0.56 7.7 ---
2 10.2 0.82 12.4 ---
1& 2 7.4 0.75 9.8 ---
1 1 0 60 5.9 0.77 7.7 95 2 3 7.7 0.77 10 59
3 4 6.1 0.77 7.8 65
4 6 10.0 0.77 13 6
1 1 0 55 16.5 1.1 15 85
2 3 13.9 1.1 12.6 79
3 4 13.6 1.1 12.4 85
4 6 9.8 1.1 8.9 57
1 1 0 50 30.3 1.7 17.8 91
2 3 41.6 1.7 24.5 76
3 4 32.2 1.7 18.9 87
4 6 17.8 1.7 10.5 82
Tm( Cl
2 1 1 0 60 10.3 0.87 11.8 74
2 9.8 1.5 6.5 71
1 & 2 9.9 0.58 17 70
21 1 & 2 1 0 60 9.3 0.66 14.1
- 2 3 9.6 0.66 14.6
3 4 9.5 0.66 14.4
4 6 7.2 0.66 10.9
=~ Number of Mismatches. 't = DH Temperature (aC). Separate experiment
These results demonstrate that the branched nucleic
acid probes can tolerate 3 and 4 mismatches with little or
no effect on performance characteristics, and can tolerate
6 mismatches with only a small decrease in performance
characteristics. Therefore branched nucleic acid probes
can be used in assay formats that require relative insen-
sitivity to mismatches. Furthermore, these data again
demonstrate the ability to include multiple AE's per probe
design (i.e., label amplification).
~.-..-.~.......:.y....~ ...... . ........,~,,...~.,,..~:.._ ,. :. .. _,...
.,:..,.... ... .. ,.... . ,.... , rt..._.. ... ..., . .... _...... .. _ ... ..
., _ . . .. . ._... ... _ _ _ _
. .. . ........ ..... :. .. . ... , . .:... . ...i.......i .:=.. . .. ....: .
' i, . ... .. t.Y... . .. . . = x . . . ..
WO 93/15102 PCT/US93/00486
~
. 2 8
Structure 2 was further evaluated by detecting
decreasing amounts of the perfectly matched target (target
1) in a full format assay as described in Example 4
(strands 1 and 2 AE labeled, 0.1 pmol each strand used;
5 target concentrations given below). The results are as
follows:
[Target] (fmol) Signal (RLU)
30 840172
10 339581
10 3 114496
1 35022
0.3 11175
0.1 3755
These data demonstrate that the branched nucleic acid
15 probe shown here is capable of detecting small quantities
of target nucleic acid in a full format assay.
Detection of Branch Duglex
The target-dependent formation of a duplex between
complementary arm regions of a branched nucle'ic acid probe
20 can be detected using techniques other than the AE-label,/,
differential hydrolysis technique discussed above. Fur-
thermore, this differential duplex formation can give rise
to a wide variety of biological or the chemical properties
which are target-dependent. The two-strand branched
25 nucleic acid probe described in FIG. 1 will be used as a
model= to discuss some examples of these properties,
including alternate modes of detection. However, it will
be readily recognized that these properties are applicable
to any number of different branched nucleic acid probe
30 configurations as long as the basic criterion that the arm
regions in question form a stable duplex only in the pres-
ence of target is met.
WO 93/15102 PCT/US93/00486
nr*cJ ~ ,,J '~'~t' 36
~..
Examole 8: Restriction Endonuclease Cleavage
In one system (FIG. 15A), arm region duplex formation
creates an active (double-stranded) restriction enzyme
cleavage site (R) removed from the target strand. Cleav-
age at this site with a restriction endonuclease can be
detected in a variety of ways. For example, one or both
of the strands can be labeled with 32P, and cleavage prod-
ucts can be detected using gel electrophoresis or other
separation techniques. Alternatively, one or both of the
strands can be labeled on one side of the cleavage site
with a capture agent (such as biotin), and the other side
of the cleavage site can be labeled with a reporter group
(e=a., 32P, AE). After cleavage, the strands are captured
(with avidin agarose, for example), and reporter group
associated with capture support indicates no cleavage,
whereas reporter group associated with the supernatant
indicates cleavage. Alternatively, a detection probe can
be designed to react with the uncleaved strand but not the
cleaved strand. This can be performed in a homogeneous or
a separation assay (the homogeneous.format'is depicted in
FIG. 15B). In one particular aspect of this system, the
cleavage site is designed to be close to the base (i.e.,
the portion near the junction) of the arm region duplex,~,
Upon cleavage with, the restriction nuclease, the arm
regions are almost entirely removed' from the 3-way
branched nucleic acid structure, thus reducing the stabil-
ity of the overall structure. If the probe regions are
designed to be stable only in the presence of intact arm
duplex, once cleavage occurs the probe regions will melt
off of the target. This will then allow uncleaved probe
strands to hybridize.with the target and start the process
over again, thus increasing assay sensitivity by cycling
multiple probes through a single target site.
Example 9: DNALRNA Polymerase Extensions
In another system (FIG. 15C), the arm regions are
designed so as to create a site for extension by a poly- ~ .. ,.
~~il.?~.~'kiiti.:' ~_.~.,..k.d ..,y~.vt..~:_., ~,. ,. . , .. ,r.' ~. _ ,,.,
.,.... .....
WO 93/15102 2 5 2 PC.'i/US93/00486
37
merase. This filled-in region can be detected by first 32P
labeling the shorter strand and then detecting any
extended primer strand using gel electrophoresis or other
separation techniques. The filled-in sequence can also be
detected using an AE-labeled probe specific for the exten-
sion product in a differential hydrolysis format. If a
restriction site is included near the base of the arm
region duplex, this system also has the potential for
cycling as described above.
ExaWle 10: Amplification
In another system (FIG. 15D) fill-in with a polymer-
ase creates an active T7 RNA promoter site. Subsequent to
fill-in, T7 RNA polymerase will transcribe multiple copies
of the template strand, thus yielding an amplification of
detectable products and thus an increase in assay sensi-
tivity. These strands can be detected, g:a., by incor-
poration of radiolabeled ribonucleotides and subsequent
separation (e.g., by precipitation), or by differential
hydrolysis of a complen-entary AE-labeled probe. If a
restriction site is included near the base of the arm
region duplex, this system also has the potential for
cycling as described above. RNA transcripts are detected.0
e.a., with an AE-labelled probe.
Euanle 11: Selective degradation
In another'system (FIG. 15E), one arm region is DNA
and the other arm region (or portions thereof) is RNA.
Formation of the arm duplex creates an active RNAse H
cleavage site. Cleavage with RNAse H can be detected as
described above for system 1. This system also has the
potential for cycling as described above.
EXamele 12: Chemical Cleavaqe
In another system (FIG. 15F), a chemical cleavage
agent (X,-such as Fe=EDTA or Cu=phenanthroline) specific
for double-stranded nucleic acid is tethered to the arm
WO 93/15102 PCT/US93/00486
~ =,..
~
U 38
region of one of the probe strands. When the arm region
duplex is formed, cleavage of the nucleic acids in the
immediate vicinity,occurs (cleavage pattern is dependent
on chemical cleaver and exact location and attachment
chemistry). This cleavage can be detected as described
above. This system also has the potential for cycling as
described above.
Any other system that discriminates between single-
and double-stranded nucleic acid is potentially applicable
to the configuration generally described above, e.g.,
specific monoclonal antibodies for double-stranded nucleic
acid.
Example 13
A specific example of the system shown in FIG. 15A is
shown in FIG. 16. This structure is the same as that
shown in F G. 2, except I that the AE label is not included.
A==EII restriction endonuclease site is engineered into
the arm regions as shown. To analyze cutting of the arm
regions target nucleic acid, strand 2 of the probe was
first labeled with 32P using the standard kinasing proto-
col., Probe was then hybridized with target using the fol-
lowing conditions: Hybrid: 0.6 pmol 32P-labeled strand 2;,~0
15=pmol strand 1;'3 pmol target strand.. Control: Same as
hybrid, except no target. As a control of enzymatic
activity, strand 2 was evaluated an exactly complemen-
tary DNA target strand. These controls will be referred
to as Linear-Hybrid and Linear-Control. The amounts used
were the same as above (0.6 pmol 32P-labeled strand.2; 3
pmol target strand). These annealing reactions were incu-
bated for 60 min at 60 C in 60 1 of 50mM Tris buffer, pH
7.9 (at 25 C), 500mM NaCl. Aliquots of each annealing
reaction were then digested with BstEII under the follow-
ing conditions: 10 1 of each reaction above diluted to
100 l with the final conditions of 50mM Tris buffer., pH
7.9 (at 25 C), 100mM NaCl, 10mM MgC121 1mM DTT. The sam-
ples were incubated with either 10, 5, 1 or 0.2 units of
WO 93/15102 PCI'/US93/00486
.v;.=~;,i
39
~stEII (New England Biolabs) for 60 min at 60 C. Aliquots
were then analyzed using standard polyacrylamide gel elec-
trophoresis (20% gel, 500V, 15mA, 2.5 hours). A standard
autoradiogram of the gel was then performed.
The results are shown in FIG. 17 and demonstrate
target-dependent cleavage of the arm region duplex at 10
and 5 units of BstEII. Furthermore, this restriction site
can be any one that is desired since the sequence of the
arm regions is independent of the target sequence. In
this manner, use of a restriction enzyme will not be
dependent on finding the correct recognition sequence in
the target to be analyzed.
ExamDle 14
A specific example of systems shown in FIGS. 15B and
15C is shown in FIG. 18. This structure contains a tem-
plate strand (strand 1) and a primer strand (strand 2).
The template strand contains a detection sequence at its
5' end as well as a T7 promoter site 3' of the detection
sequence (see FIG.. 18). Upon hybridization of both
strands to the target, the. arm regions form a stable
duplex, and the 3' end of strand 2 becomes an active
primer extension site for a polymerase enzyme. The enzynt,ee
fi=lls-in the complement to the template strand, which can
then be denatured and detected using the AE-labeled detec-
tion oligomer shown in FIG. 18. Fill-in with the poly-
merase also creates an active (double-stranded) T7 pro-
moter region with the same detection sequence template
region. A T7 RNA polymerase enzyme can then transcribe
multiple RNA copies off of the template, which are also
detected with the AE-labeled detection oligomer.
The probe was first hybridized with the target strand
by incubating 0.3 pmol of strand 1, 0.9 pmol of strand 2
and 0.1 pmol of the target strand in 50mM Tris buffer, pH
7.9 (@ 25 C), 0.5M NaCl (10 1 final volume) at 60 C for 60
minutes (Control sample contained no target). This sample
was then diluted to 50 1 for extension with DNA polymerase
. ..
~.41'~4 -. t .'1: t ~'lT . t s '. ~ +. : c : - , .. . ~, ~,; . . .,: I ~. ie
'x=:..i.. . . , . . .... . .:4..., .. .. .. . . .. .......... ..... . . .. _ .
. __ -
._ _U_ . , ... .......,.:J. lL. J../t.G..... ....,_....:. ...y. ......,.. .',
xn.:.. _ .' .. .', -=. .. .'.,... : :.'..:"
CA 02128530 2003-10-29
73091-27
under the following conditions: 20mM Tris buffer, pH 7.9
(at 25 C), 0.15M NaC1,.3mM MqSO4, 1% TX-100, 1mM DTT, 2mM
dATP, 2mM dTTP, 2mM dGTP, 2mM dCTP and 4 units of Taq
polymerase (Cetus Corp.). The reaction was incubated at
5 60 C for one hour. A 5 l aliquot of the sample was then
mixed with 25 1 of 6% lithium lauryl suifate,. 60mM sodium
phosphate buffer, pH 6.8, 2mM EDTA and ~2mM EGTA and 20 1
of water. This sample was incubated at 95 C for 5 min-
utes, cooled to 60 C or lower, and then= mixed with 50g1 of
10 200mM lithium succinate buffer, pH 5.2, 17% lithium lauryl
sulfate, 2mM EDTA, 2mM EGTA and 0.1 pmol of AE-labeled
detection oligomer. This reaction mixture was incubated.
for 60 minutes at 60 C. Next, 300 l of 0.15M sodium
borate buffer, pH 7.6,,containing 5% Triton MX-100 deter-
15 gent was added, and the sample was incubated at 60 C for
10 minutes. The chemiluminescence was measured in a lumi-
nometer (LEADER I, Gen-Probe, CA) by the automatic injec-
tion of 200 1 of 1mM HNO3, 0.1% H2021 then 200 1 of 1N NaOH,
2% Zwittergent followed by measurement of signal. for 2
20 seconds.
For transcription with T7 polymerase, 5g1 of the
extension mixture was added to 45 1 of a transcription
mixture such that the final conditions were as follows:
40mM Tris buffer, pH 7.9 (at 25 C), 6mM MgC12, 2mM spermi-
25 dine, 10mM DTT, 2.5mM rCTP, 2.5mM rUTP, 6.5mM rATP, 6.5mM
rGTP and 400 uinits of T7 RNA polymerase (United States
Biochemical Corporation). A 5 1 aliquot of this sample
was assayed using the- AE.-labeled detection oligomer as
described above.
30 The following results were obtained:
AE-probe assay (RLUL
Activity Hybrid Control
Taq polymerase 74,125 1,083
T7 RNA polymerase 641,753 97,218
~5+1d~606:% cii~=+1::'M..t.'b?!r::[J.S{~~'LY't:~:=r~.;~J~ei::c4~d:?!4!'~7,i::.
r.k'~;s"~."!3~~.f + ~'r ~yy10=.?. ..0C~ 5 =:r,..- ~' . '':'' . .. . . .
+s..lJ. h.
WO 93/ 15102 PC,'I /US93/00486
~~~8 -5'~
41
These data demonstrate that sites for Tag polymerase
and T7 RNA polymerase can be created separate from the
target region, yet the activity of these sites is modu-
lated by hybridization to target.
Example 15:
A specific example of the general configuration shown
in FIG. 6E is shown in FIG. 21. The target strand is a
synthetic 38mer oligomer; the probe strands form a 4-way
junction with the target strand as shown. All oligomers
were synthesized as described in Example 1 above. Hybri-
dization characteristics of the structures shown in FIGS.
22A-221 (various schematic representations of the struc-
ture shwon in FIG. 21) were evaluated using PH analysis
protocols described in Example 1; -all operations were
performed at 50 C. The amounts of the different strands
used were always the same (even if not all the strands
were used) and were as follows: target strand - 1.5 pmol;
strand 1 - 0.1 pmol; strand 2 - 6:5 pmol; strand 3 1.0
pmol. The following results were obtained:
Stucture Hydrolysis -
(Fig. Number) Half-life (min.)
22A 37.0
22B 0.54
22C 1.7
22D 0.72
22E 0.69
22F 0.60
22G 1.7
22H 42.7
221 0.55
These data demonstrate that-the AE is not protected
from ester hydrolysis in the absence of target but is pro-
tected from ester hydrolysis in the presence of target
'(and the other probe strands). In this particular exam-
ple, all four strands must be present to form a stable
duplex between the arm regions of probe strands 1 and 2.
WO 93/15102 PCT/US93/00486
c.,~;
k ,..
42
Taraet Independent Probe Amplification
In another configuration, some of the duplexes formed
between complementary arm regions are formed independent
of target (as long as at least one such duplex is formed
only in the presence of target). Some examples of this
configuration are shown in FIGS. 19A-19B. One of the
utilities of this configuration is label amplification,
since AE or another label can be placed in multiple
regions of a "network" of branched nucleic acid struc-
tures. Furthermore, when AE is the label, this confi-
guration allows for a universal detection complex that
could be used for any desired target site. This structure
will become associated with the target molecule when the
two (or more) probe regions hybridize to target, thus
stabilizing the "anchor" junction (as depicted in FIGS.
19A and 19B). The target with this associated label
amplification network will then typically have to be
separated from unassociated network by some heterogeneous
method (e=a:, sandwich assay, hydroxyapatite capture of
target nucleic acid). The number of junctions formed with
target can be unlimited (at least up to 5, or even 10, and
depending on the size of the probe, can be several thou-
sands, so long as the hybridized probes do not precipitate+
from solution),.as can the formation of a detection net-
work which may associate with probe before or after hybri-
dization to target.
In one case of the configuration discussed above, all
the junctions could form in a target-independent manner.
Some examples of this configuration are shown in FIGS.
20A-20C. This would allow for label amplification, using
the branched nucleic acid structures to create networks,
thereby allowing the incorporation of multiple labels per
target site. This strategy can be used with a probe that
forms a nucleic acid junction with the target strand (this
.35, junction is target-dependent in the sense that the full
junction cannot form in the absence of target, but the
duplex formed between associated arm regions is target-
WO 93/15102 PCT/US93/00486
t c (h~ ~
i ~ {~7 lJ l fJ .1
43
independent in that they form even in the absence of
target), or a probe that does not form a nucleic acid
junction with the target strand.
Therapeutic Probes
In addition to the detection and quantitation of
nucleic acid targets, the branched nucleic acid probe
configurations described herein can also be utilized in a
variety of therapeutic applications. For example, the
general configuration shown in FIG. 15, system 3, could be
utilized by designing the probe regions to be specific
for, e.g., an infectious organism, or specific cancer
cells, and the arm regions to code for a biologically
active molecule. The probes will hybridize with the
target site, the template strand filled in by endogenous
polymerases (this step might be unnecessary), producing an
active coding region which will then generate (again via
endogenous activities) a biologically active molecule,
such. as an inhibitor of an essential transcription or
translation factor, a peptide or other small molecule that
is toxic to the organism or cell, or an antigen that ren-
ders the cell susceptible to removal by the immune system.
- Alternatively, the arm regions may form a double;.#
stranded RNA region in a target-dependent manner, which in
turn stimulates,interferon activity. As another example,
the junction formed with a target nucleic acid can be
designed to form a site such that (at least) the target
strand is cleaved by an endogenous resolvase enzyme. The
probe will target a site where this cleavage would be
lethal to the organism, or cell.
As another example, the duplex arm regions may create
a recognition site for an endogenous nuclease that binds
to this recognition site but cleaves at a distal site
located on the target strand, with this cleavage leading
to death of the organism, or cell. Alternatively, the arm
region duplex will create a recognition site that competes
for the binding of an essential endogenous regulation pro-
WO 93/ 151 Q2 PCT/US93/00486
44
tein, or interferes with the assembly of an essential
regulation complex in a sequence specific manner. Fur-
ther, various combinations of probes could be designed to ,
form stable branch points with an essential region of the
target nucleic acid, thus blocking binding of sequence
specific processing factors and thereby inhibiting
replication.
Other embodiments are within the following claims.
~.f
