Note: Descriptions are shown in the official language in which they were submitted.
CA 02385853 2002-03-27
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Design of high affinity RNase H recruiting oligonucleotide
Field of Invention
The present invention relates to the field of bicyclic DNA analogues which are
useful for
designing oligomers that forms high affinity duplexes with complementary RNA
wherein
said duplexes are substrates for RNase H. The oligonucleotides may be
partially or fully
composed of bicyclic DNA analogues.
Background of the invention
The term "antisense" relates to the use of oligonucleotides as therapeutic
agents. Briefly,
an antisense drug operates by binding to the mRNA thereby blocking or
modulating its
translation into protein. Thus, antisense drugs may be used to directly block
the synthesis
of disease causing proteins. It may, of course, equally well be used to block
synthesis of
normal proteins in cases where these participate in, and aggravate a
pathophysiological
process. Also, it ought to be emphasised that antisense drugs can be used to
activate
genes rather that suppressing them. As an example, this can be achieved by
blocking the
synthesis of a natural suppressor protein.
Mechanistically, the hybridising oligonucleotide is thought to elicit its
effect by either cre-
ating a physical block to the translation process or by recruiting a cellular
enzyme (RNase
H) that specifically degrades the mRNA part of the mRNA/antisense
oligonucleotide du-
plex.
Not unexpectedly, oligonucleotides must satisfy a large number of different
requirements
to be useful as antisense drugs. Importantly, the antisense oligonucleotide
must bind with
high affinity and specificity to its target mRNA, must have the ability to
recruit RNase H,
must be able to reach its site of action within the cell, must be stable to
extra - and intra-
cellular nucleases both endo- and exo-nucleases, must be non-toxic/minimally
immune
stimulatory, etc.
Natural DNA only exhibit modest affinity for RNA and fall short on a number of
the other
critical characteristics, especially nuclease resistance. Hence, a significant
effort has been
invested to identify novel analogues with improved antisense properties. In
particular the
search has focused on identifying novel analogues, which combine an increased
affinity
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2
for complementary nucleic acids with the RNase H recruiting ability of natural
DNA. Both
of these properties have been demonstrated to correlate in a strongly positive
manner
with biological activity. Of the vast number of analogues that have emerged
from this
work, only few retain the ability to recruit RNase H and very few provide
useful increases
in affinity. Sadly, those that do provide a useful increase in affinity fail
to recruit RNase H.
In the face of these results the field have turned to mixed backbone
oligonucleotides as a
means to provide higher potency antisense drugs, i.e. antisense molecules that
operates
by a two fold mechanism of action 1 ) high affinity mediated translational
arrest at the ribo-
somal level and 2) activation of RNase H. These molecules (termed gab-mers)
typically
comprise a central region of at least six contiguous, low affinity
phosphorothioates (RNase
H recruiting analogues) flanked by stretches of high affinity analogues (non
RNase H re-
cruiting analogues) that enhance the ability to promote translational arrest.
Although ex-
pected to out-perform current phosphorothioate antisense drugs, the gab-mers
are not
considered the ideal antisense molecules. Amongst their weaknesses is the
requirement
for a rather fixed design and the presence of high and tow affinity domains
within the
molecule, which may compromise biological activity.
The enzyme RNase H selectively binds to heterogeneous DNA/RNA duplexes and de-
grades the RNA part of the duplex. Homogeneous DNA/DNA and RNA/RNA duplexes,
which only differs molecularly from the DNA/RNA duplex at the 2' position
(DNA/DNA: 2'-
H/2'-H; RNA/RNA: 2'-OH/2'-OH and DNA/RNA: 2'-H/2'-OH) are not substrates for
the
enzyme. This suggests that either the molecular composition at the 2' position
itself or the
structural feature it imposes on the helix is vital for enzyme recognition.
Consistent with
this notion, all 2'-modified analogues that have so far been reported to
exhibit increased
affinity have lost the ability to recruit RNase H.
Detailed description of the invention.
Locked Nucleic Acid (LNA) is a novel, nucleic bicyclic acid analogue in which
the 2'- and
4' position of the furanose ring are linked by an O-methylene (oxy-LNA), S-
methylene
(thio-LNA) or NHZ-methylene moiety (amino-LNA). This linkage restricts the
conforma-
tional freedom of the furanose ring and leads to an increase in affinity which
is by far the
highest ever reported for a DNA analogue (WO 99/14226).
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3
Despite the fact that the modification in LNA involves the 2'-position we have
found that
the activity of RNase H is not dependent on a contiguous stretch of DNA or
phosphoro-
thioated bases when oxy-LNA is used as a component of the oligonucleotide. In
fact, we
have found that oligonucleotides composed entirely of oxy-LNA are able to
recruit RNase
H. Oxy-LNA oligonucleotides thus constitutes the first ever DNA analogue to
display the
long sought after combination of very high affinity and ability to recruit
RNase H. The im-
plications are that oxy-LNA by itself may be used to construct novel antisense
molecules
with enhanced biological activity. Alternatively, oxy-LNA may be used in
combination with
non-oxy-LNA, such as standard DNA, RNA or other analogues, e.g. thio-LNA or
amino-
LNA to create high affinity, RNase H recruiting antisense compounds without
the need to
adhere to any fixed design.
An "oxy-LNA monomer" is defined herein as a nucleotide monomer of the formula
la
R5 R5*
P B
R4*,,,,,. ~.", R~* la
Ra . . R2
R3* R2*
wherein X is oxygen; B is a nucleobase; R' , R2, R3, R5 and RS' are hydrogen;
P desig-
nates the radical position for an internucleoside linkage to a succeeding
monomer, or a 5'-
terminal group, R3 is an internucleoside linkage to a preceding monomer, or a
3'-terminal
group; and RZ and R4' together designate -O-CHZ- where the oxygen is attached
in the 2'-
position.
The term "nucleobase" covers the naturally occurring nucleobases adenine (A),
guanine
(G), cytosine (C), thymine (T) and uracil (U) as well as non-naturally
occuring nucleo-
bases such as xanthine, diaminopurine, 8-oxo-N6-methyladenine, 7-
deazaxanthine, 7-
deazaguanine, N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-
methylcytosine,
5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-
hydroxy-5-
methyl-4-triazolopyridin, isocytosine, isoguanin, inosine and the "non-
naturally occurring"
nucleobases described in Benner et al., U.S. Pat No. 5,432,272 and Susan M.
Freier and
Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol. 25, pp 4429-4443. The
term "nu-
cleobase" thus includes not only the known purine and pyrimidine heterocycles,
but also
heterocyclic analogues and tautomers thereof. It should be clear to the person
skilled in
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4
the art that various nucleobases which previously have been considered "non-
naturally
occurring" have subsequently been found in nature.
A "non-oxy-LNA" monomer is broadly defined as any nucleoside (i.e. a glycoside
of a het-
erocyclic base) which is not itself an oxy-LNA but which can be used in
combination with
oxy-LNA monomers to construct oligos which have the ability to bind sequence
specifi-
cally to complementary nucleic acids. Examples of non-oxy-LNA monomers include
2'-
deoxynucleotides (DNA) or nucleotides (RNA) or any analogues of these monomers
which are not oxy-LNA, such as for example the thio-LNA and amino-LNA
described by
Wengel and coworkers (Singh et al. J. Org. Chem. 1998, 6, 6078-9, and the
derivatives
described in Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research,
1997, vol
25, pp 4429-4443.
It should be understood that the incorporation of non-oxy-LNA monomers) into
an oxy-
LNA oligo may change the RNAseH recruiting characteristics of the oxy-LNAlnon-
oxy-
LNA chimeric oligo. Thus, depending on the number and type of non-oxy-LNA mono-
mers) used, and the position of these monomers in the resulting oxy-LNA/non-
oxy-LNA
chimeric oligo, the chimera may have an increased, unaltered or decreased
ability to re-
cruit RNAsdeH as compared to the corresponding all oxy-LNA oligo.
As mentioned above, a wide variety of modifications of the deoxynucleotide
skeleton can
be contemplated and one large group of possible non-oxy-LNA can be described
by the
following formula I
R5 R5.
P
Ra. R~.
R Rs* R2 R2
wherein X is -O-; B is selected from nucleobases; R'~ is hydrogen;
P designates the radical position for an internucleoside linkage to a
succeeding monomer,
or a 5'-terminal group, such internucleoside linkage or 5'-terminal group
optionally includ-
ing the substituent R5, RS being hydrogen or included in an internucleoside
linkage,
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R3* is a group P* which designates an internucleoside linkage to a preceding
monomer, or
a 3'-terminal group;
one or two pairs of non-geminal substituents selected from the present
substituents of R2,
5 R2', R3, R4', may designate a biradical consisting of 1-4 groups/atoms
selected from
-C(RaRb)-, -C(Ra)=C(Ra)-, -C(Ra)=N-, -O-, -S-, -SOz-, -N(Ra)-, and >C=Z,
wherein Z is selected from -O-, -S-, and -N(Ra)-, and Ra and Rb each is
independ-
ently selected from hydrogen, optionally substituted C,_6-alkyl, optionally
substi-
tuted C2_6-alkenyl, hydroxy, C,_6-alkoxy, CZ_6-alkenyloxy, carboxy, C,_s-
alkoxy-
carbonyl, C,_6-alkylcarbonyl, formyl, amino, mono- and di(C,_6-alkyl)amino,
car-
bamoyl, mono- and di(C,_6-alkyl)-amino-carbonyl, amino-C,_6-alkyl-
aminocarbonyl,
mono- and di(C,_6-alkyl)amino-C,_s-alkyl-aminocarbonyl, C,_6-alkyl-
carbonylamino,
carbamido, C,_6-alkanoyloxy, sulphono, C,_6-alkylsulphonyloxy, nitro, azido,
sul-
phanyl, C,_6-alkylthio, halogen, photochemically active groups,
thermochemically
active groups, chelating groups, reporter groups, and ligands,
said possible pair of non-geminal substituents thereby forming a monocyclic
entity to-
gether with (i) the atoms to which said non-geminal substituents are bound and
(ii) any
intervening atoms; and
each of the substituents Rz, Rzt, R3, R4 which are present and not involved in
the possible
biradical is independently selected from hydrogen, optionally substituted C,_6-
alkyl, op-
tionally substituted CZ_6-alkenyl, hydroxy, C,_s-alkoxy, CZ_6-alkenyloxy,
carboxy, C,_6-
alkoxycarbonyl, C,_6-alkylcarbonyl, formyl, amino, mono- and di(C,_6-
alkyl)amino, car-
bamoyl, mono- and di(C,_6-alkyl)-amino-carbonyl, amino-C,_6-alkyl-
aminocarbonyl, mono-
and di(C,_6-alkyl)amino-C,~-alkyl-aminocarbonyl, C,_6-alkyl-carbonylamino,
carbamido,
C,_6-alkanoyloxy, sulphono, C,_6-alkylsulphonyloxy, vitro, azido, sulphanyl,
C,_6-alkylthio,
halogen, photochemically active groups, thermochemically active groups,
chelating
groups, reporter groups, and ligands;
and basic salts and acid addition salts thereof;
with the proviso the monomer is not oxy-LNA.
Particularly preferred non-oxy-LNA monomers are 2'-deoxyribonucleotides,
ribonucleo-
tides, and analogues thereof that are modified at the 2'-position in the
ribose, such as 2'-
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6
O-methyl, 2'-fluoro, 2'-trifluoromethyl, 2'-O-(2-methoxyethyl), 2'-O-
aminopropyl, 2'-O-
dimethylamino-oxyethyl, 2'-O-fluoroethyl or 2'-O-propenyl, and analogues
wherein the
modification involves both the 2'and 3' position, preferably such analogues
wherein the
modifications links the 2'- and 3'-position in the ribose, such as those
described by Wen-
gel and coworkers (Nielsen et al., J. Chem. Soc., Perkin Trans. 1, 1997, 3423-
33, and in
WO 99/14226), and analogues wherein the modification involves both the 2'and
4' posi-
tion, preferably such analogues wherein the modifications links the 2'- and 4'-
position in
the ribose, such as analogues having a -CH2-S- or a -CHZ-NH- or a -CH2-NMe-
bridge
(see Wengel and coworkers in Singh et al. J. Org. Chem. 1998, 6, 6078-9).
Although,
non-oxy-LNA monomers having the (3-D-ribo configuration are often the most
applicable,
further interesting examples (and in fact also applicable) of non-oxy-LNA are
the stereoi-
someric of the natural (3-D-ribo configuation. Particularly interesting are
the a-L-ribo, the (3-
D-xylo and the a-L-xylo configurations (see Beier et al., Science, 1999, 283,
699 and Es-
chenmoser, Science, 1999, 284, 2118), in particular those having a 2'-4' -CH2-
S-, -CHZ-
NH-, -CHZ-O- or -CH2-NMe- bridge (see Wengel and coworkers in Rajwanshi et
al.,
Chem. Commun., 1999, 1395 and Rajwanshi et al., Chem. Commun., 1999,
submitted)
In the present context, the term "oligonucleotide" which is the same as
"oligomer" which is
the same as "oligo" means a successive chain of nucleoside monomers (i. e.
glycosides of
heterocyclic bases) connected via internucleoside linkages. The linkage
between two
successive monomers in the oligo consist of 2 to 4, preferably 3, groups/atoms
selected
from -CH2-, -O-, -S-, -NR"-, >C=0, >C=NR", >C=S, -Si(R")2-, -SO-, -S(O)2-, -
P(O)2-,
-PO(BH3)-, -P(O,S)-, -P(S)2-, -PO(R")-, -PO(OCH3)-, and -PO(NHR")-, where R"
is se-
lected form hydrogen and C,.~-alkyl, and R" is selected from C,.6-alkyl and
phenyl. Illu-
strative examples of such linkages are -CH2-CHZ-CH2-, -CHz-CO-CHZ-, -CHZ-CHOH-
CHZ-,
-O-CHZ-O-, -O-CHz-CH2-, -O-CHZ-CH= (including RS when used as a linkage to a
suc-
ceeding monomer), -CHz-CH2-O-, -NR"-CHZ-CHZ-, -CH2-CH2-NR"-, -CH2-NR"-CH2-,
-O-CH2-CHZ-NR"-, -NR"-CO-O-, -NR"-CO-NR"-, -NR"-CS-NR"-, -NR"-C(=NR")-NR"-,
-NR"-CO-CHZ-NR"-, -O-CO-O-, -O-CO-CH2-O-, -O-CH2-CO-O-, -CHZ-CO-NR"-, -O-CO-
NR"-, -NR"-CO-CHZ-, -O-CH2-CO-NR"-, -O-CH2-CH2-NR"-, -CH=N-O-, -CHZ-NR"-O-,
-CH2-O-N= (including RS when used as a linkage to a succeeding monomer), -CHz-
O-
NR"-, -CO-NR"-CH2-, -CHZ-NR"-O-, -CH2-NR"-CO-, -O-NR"-CHz-, -O-NR"-, -O-CH2-S-
,
-S-CH2-O-, -CH2-CH2-S-, -O-CHZ-CH2-S-, -S-CHZ-CH= (including R5 when used as a
link-
age to a succeeding monomer), -S-CHZ-CHZ-, -S-CHZ-CHZ-O-, -S-CHZ-CH2-S-, -CH2-
S-
CHZ-, -CH2-SO-CHZ-, -CH2-S02-CHZ-, -O-SO-O-, -O-S(O)2-O-, -O-S(O)2-CHZ-, -O-
S(O)2-
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NR"-, -NR"-S(O)2-CH2-, -O-S(O)2-CH2-, -O-P(O)2-O-, -O-P(O,S)-O-, -O-P(S)2-O-,
-S-P(O)2-O-, -S-P(O,S)-O-, -S-P(S)2-O-, -O-P(O)2-S-, -O-P(O,S)-S-, -O-P(S)2-S-
,
-S-P(O)2-S-, -S-P(O,S)-S-, -S-P(S)Z-S-, -O-PO(R")-O-, -O-PO(OCH3)-O-, -O-PO-
(OCH2CH3)-O-, -O-PO(OCH2CHzS-R)-O-, -O-PO(BH3)-O-, -O-PO(NHR")-O-, -O-P(O)2-
NR"-, -NR"-P(O)Z-O-, -O-P(O,NR")-O-, -CHZ-P(O)2-O-, -O-P(O)2-CHZ-, and -O-
Si(R")2-O-;
among which -CH2-CO-NR"-, -CHZ-NR"-O-, -S-CH2-O-, -O-P(O)2-O-, -O-P(O,S)-O-,
-O-P(S)2-O-, -NR"-P(O)2-O-, -O-P(O,NR")-O-, -O-PO(R")-O-, -O-PO(CH3)-O-, and
-O-PO(NHR")-O-, where R" is selected form hydrogen and C,~-alkyl, and R" is
selected
from C,_6-alkyl and phenyl, are especially preferred. Further illustrative
examples are
given in Mesmaeker et. al., Current Opinion in Structural Biology 1995, 5, 343-
355 and
Susan M. Freier and Karl-Heinz Altmann, Nucleic Acids Research, 1997, vol 25,
pp 4429-
4443. The left-hand side of the internucleoside linkage is bound to the 5-
membered ring
as substituent P at the 3'-position, whereas the right-hand side is bound to
the 5'-position
of a preceding monomer.
The term "succeeding monomer" relates to the neighbouring monomer in the 5'-
terminal
direction and the "preceding monomer" relates to the neighbouring monomer in
the 3'-
terminal direction.
Monomers are referred to as being "complementary" if they contain nucleobases
that can
form hydrogen bonds according to Watson-Crick base-pairing rules (e.g. G with
C, A with
T or A with U) or other hydrogen bonding motifs such as for example
diaminopurine with
T, inosine with C, pseudoisocytosine with G, etc.
When the modified oxy-LNA oligo contain at least two non-oxy-LNA monomers
these may
contain the same or different nucleobases at the 1'-position and be identical
at all other
positions or they may contain the same or different nucleobases at the 1'-
position and be
non-identical at at least one other position.
Accordingly, the present invention describes a method for degrading RNA in-
vivo (in a cell
or organism) or in-vitro by providing a high affinity oligonucleotide which
activates
RNaseH when the high affinity oligonucleotide is hybridised to a complementary
RNA tar-
get sequence, said high affinity oligonucleotide may consist of oxy-LNA
monomers exclu-
sively.
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8
Alternatively, the high affinity oligonucleotide may also consist of both oxy-
LNA and non-
oxy-LNA monomers, in this case the high affinity oligonucleotide contains at
the most five,
e.g. 4, e.g. 3 , e.g. 2 contiguous non-oxy-LNA monomers at any given position
in the oli-
gonucleotide, e.g. said high affinity oligonucleotide consists of both oxy-LNA
and non-oxy-
LNA monomers, wherein none of the non-oxy-LNA monomers are located adjacent to
each other.
The high affinity oligonucleotide may also contain one or more segments of
contigous
non-oxy-LNA monomers. For instance, a stretch of contigous non-oxy-LNA
monomers
may be located in the centre of the oligonucleotide and with flanking segments
consisting
of oxy-LNA monomers. Alternatively the stretch of contigous non-oxy-LNA
monomers may
be located at either or both ends. Also, the oxy-LNA segement(s) may be either
contigous
or interrupted by 1 or more non-oxy-LNA monomers. Also, the high affinity
oligonucleotide
may comprise more than one type of internucleoside linkage such as for example
mixes
of phosphordiester and phosphorothioate linkages.
The resulting high affinity oligo containing oxy-LNA monomers and/or non-oxy-
LNA mono-
mers can thus be characterized by the general formula
5'-(X,~,Y~XP]q 3'
X is oxy-LNA and Y is non-oxy-LNA, wherein m and p are integers from 0 to 30,
n is an
integer from 0 to 3 and q is an integer from 1 to 10 with the proviso that the
sum of m+n+p
multiplied with q is in the range of 6-100, such as 8, e.g. 9, e.g. 10, e.g.
11, e.g. 12, e.g.
13, e.g. 14, e.g. 15, e.g. 16, e.g. 17, e.g. 18, e.g. 19, e.g. 20, e.g. 21,
e.g. 22, e.g. 23, e.g.
24, e.g. 25, e.g. 26, e.g. 27, e.g. 28, e.g. 29, e.g. 30, e.g. 35, e.g. 40,
e.g. 45, e.g. 50, e.g.
60, e.g. 70, e.g. 80, e.g. 90, such as 100.
The present invention provides oligos which combine high affinity and
specificity for their
target RNA molecules with the ability to recruit RNAseH to an extend that
makes them
useful as antisense therapeutic agents. The oligos may be composed entirely of
oxy-LNA
monomers or they may be composed of both oxy and non-oxy-LNA monomers.
When both oxy-LNA and non-oxy-LNA monomers) are present in the oligo, the
RNAseH
recruiting characteristics of the chimeric oligo may be similar to, or
different from, that of
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9
the corresponding oxy-LNA oligo. Thus, in one aspect of the invention, non-oxy-
LNA
monomers) is/are used in such a way that they do not change the RNAseH
recruting
characteristics of the oxy-LNA/non-oxy-LNA chimeric oligo compared to the
correspond-
ing all oxy-LNA oligo. In another aspect of the invention the non-oxy-LNA
monomers)
is/are used purposely to change the RNAseH recruiting characteristics of an
oxy-LNA
oligo, either increasing or decreasing its efficiency to promote RNAseH
cleavage when
hybridised to its complementary RNA target compared to the corresponding all
oxy-LNA
oligo.
When both oxy-LNA and non-oxy-LNA monomers) is/are present in the oligo, the
ability
of the chimeric oligo to discriminate between its complementary target RNA and
target
RNAs containing one or more Watson-Crick mismatches may be different from the
ability
of the corresponding all oxy-LNA oligo to discriminate between its matched and
mis-
matched target RNAs. For instance, the ability of an oxy-LNA oligo to
discriminate be-
tween a complementary target RNA and a single base mismatched target RNA can
be
enhanced by incorporating non-oxy-LNA monomer(s), such as for instance DNA,
RNA,
thio-LNA or amino-LNA, either at, or close to, the mismatched position as
described in
applicant's Danish patent application entitled "Metod of increasing the
specificity of oxy-
LNA oligonuclotides" filed on the same day as the present application. Thus,
in another
aspect of the invention non-oxy-LNA monomers) is/are used purposely to
construct an
oxy-LNA/non-oxy-LNA oligo which exhibit increased specificity but unaltered
RNAseH re-
cruiting characteristics compared to the corresponding all oxy-LNA oligo. In
another as-
pect of the invention the non-oxy-LNA monomers) is/are used purposely to
construct an
oxy-LNA/non-oxy-LNA oligo which exhibit both increased specificity and altered
RNAseH
recruiting characteristics compared to the corresponding all oxy-LNA oligo
Additionally, the oligonucleotide of the present invention may be conjugated
with com-
pounds selected from proteins, amplicons, enzymes, polysaccharides,
antibodies, hap-
tens and peptides.
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Examples
Example 1: LNA containing oligonucleotides recruit RNase H
Two 41-mer oligonucleotides, that make up a linearised double-stranded
template for
subsequent T7 polymerise run-off transcription, were used to obtain target RNA
corre
5 sponding to the following 15mer oligonucleotides:
DNA control; 5'- gtgtccgagacgttg-3'
phosphorothioate control; 5'-gtgtccgagacgttg-3'
LNA gab-mer; 5'-GTGTccgagaCGTTG-3' (LNA in capital letters, DNA is small
letters) and
10 LNA-mix-mer; 5'-gTgTCCgAgACgTTg-3' (LNA in capital letters, DNA is small
letters)
In the 5'end of the sense template strand, the promoter sequence for T7
polymerise rec-
ognition and initiation of transcription were contained, followed by the DNA
sequence
coding for the target-RNA sequence. The two complementary oligonucleotides
were
heated to 80°C for 10min to produce the linearised double-strand
template. A 201 in vitro
transcription reaction containing 500~M each of ATP, GTP and CTP, 12pM of UTP,
ap-
prox. 50pCi of a-32P UTP, 1 x transcription buffer (Tris-HCI, pH 7.5), 10mM
dithiotretiol,
1 % BSA, 20 U of RNasin ribonuclease inhibitor, 0.2 ~I template and 250 units
T7 RNA
polymerise. The inclusion of RNasin inhibitor was to prevent degradation of
the target-
RNA from ribonucleases. The reactions were carried out at 37°C for 2h
to produce the
desired 24mer 32U-labelled RNA run-off transcript. For target RNA
purification, 1.51 (1.5
Units) of DNase I was added to the RNA which was resolved in a 15%
polyacrylamide gel
containing 7M urea and the correctly sized fragment was excised from the gel,
dispensed
in elution buffer (0.1 % SDS, 0.5M ammonium acetate, 1 OmM Mg-acatate) and
incubated
at room temperature overnight. The target RNA sequence was then purified via
ethanol
precipitation, the supernatants filtered through a Millipore (0.45m) and
collected by etha-
nol precipitation. The pellets were diluted in TE-buffer and subsequently
subjected to
RNAse H digestion assay. Herein, the decrease of intact substrate, i.e. the 24-
mer a-32P
UTP labelled target RNA sequence, was assayed over time as follows. The
reactions
were carried out in a total volume of 110p.1 and contained (added in the order
mentioned):
1 x nuclease-free buffer (20mM Tris-HCI, pH 7.5, 40mM KCI, 8mM MgCl2, 0.03
mgiml
BSA), 10mM dithiotretiol, 4% glycerol, 100nM of oligonucleotide, 3 Units
RNasin inhibitor,
labelled target RNA strand and 0.1 U of RNase H. An excess of oligonucleotide
was
added to each reaction to ensure full hybridisation of the RNA target
sequences. Two
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11
negative controls were also included and were prepared as above but (1)
without any oli-
gonucleotide, or (2) without RNase H added to the reaction mixture. All the
reactions were
incubated at 37°C. At time points 0, 10, 20, 40 and 60 min., 10u1
aliquots were taken and
immediately added to ice-cold formamide loading buffer to quench the reaction
and stored
at -20°C. The samples were heated to 85°C for 5 min. prior to
loading and running on a
15% polyacrylamide gel containing 7M urea. The gels were vacuum dried and
exposed to
autoradiographic films over night and subsequently subjected to densitometric
calcula-
tions using the Easy Win imaging software (Hero Labs). The volume density of
intact tar-
get RNA were calculated in each lane with correction for background. The
volume density
for the time zero sample was set as reference value for each incubation.
Relative values
for the other time-points samples in the corresponding incubation were
calculated based
on these reference values.
Brief description of figures
Figure 1 shows the results of the RNase H experiments. As expected the control
DNA and
phosphorothioate oligonucleotides both recruit RNAse H very efficiently. Also,
as expect-
ed the LNA oligonucleotide which contains a contiguous stretch of six DNA
monomers in
the middle (LNA gab-mer) recruits RNAse H efficiently. Surprisingly, however,
the LNA
mix-mer which contains only single DNA monomers interdispersed between LNA
mono-
mers also recruits RNAse H. We conclude that the activity of RNase H is not
contingent
on a contiguous stretch of DNA or phosphorothioated bases when LNA is used as
a com-
ponent of the oligonucleotide.
