Note: Descriptions are shown in the official language in which they were submitted.
WO91/18997 ~CTtUS91~3680
--1~
SEQUENCE-SPECIFIC NONPHOTOACTIVATED CROSSLINKING
AGENTS ~HICH BIND ~O ~H~ MAJOR GROOVE OF DUPLEX DNA
Technical Field
The invention relates generally to compositions
use~ul in "an~isense" therapy and diagnosis. More
particularly, it concerns compositions which are capable
of binding in a sequence-specific manner to the major
groove o~ nucleic acid duplexes and f orming co~alent
bonds with one or both strands of the duplex.
Back~round ~rt
"Antisense" therapies are generally understood
to be thos~ which target specific nucleotide sequences
associated with a disease or o~her unde~irable condition.
While the term "antisense" appears super~icially to refer
specifically to the well-known A-T and G-C
complementarity responsible for hybridization of a
"sense" strand of DNA, ~or example, to its "antisense"
strand, this term, as applied to ~he ~echnology, has come
to ~e underqtood to include any mechanism for interfering
with those aspects of the disease or condition which are
mediated by nucleic acids. Thus, in addition ko
utilizing reagents which presumably hybridize by virtue
of basepair co~plementarity to single-stranded forms such
~:~30 as mRNA or separated strands of DNA duplexes, materials
.`which destroy or interfere with the function of nucleic
acid duplexes are also e~fective.
~The invention described below relates directly
::to this aspect of "antisense" therapy (and diagnosis).
The compositions and methods useful in the invention
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target the major groove of nucleic acid duplexes in
sequence dependent manner. In order to distinguish
targeted duplexes from those which are indigenous to the
subject or which otherwise are not desired to be
S affected, this binding must be sequence specific.
Xt is now known that sin~le-stranded
oli~onucleotides are capable of sequence-specific binding
to the major groove in a duplex according to rules which
haYe been reported, for example, by MosPr and Dervan,
Science (1987) 238:645-650. In this report, sequence-
specific recognition was used to associate homopyrimidine
derivatized EDTA with the major groove and effect
cleavage of the double helix. Lesser degrees of sequence
specificity have been designed into nonoligonucleotide
molecules such as peptides, as reported by Dervan, P.B.,
Science (1986) 232:464-471 and by Baker and Dervan, J Am
Che~ Soc (1989) 111:2700-2712. The sequence-specific
reagent in this pair of reports, however, resides in the
minor groove of a DNA double helix.
Peptides which associate specifically with
sequences in double helices are also reported by Sluka,
J.P., et al., Science (1987) 238:1129-1132. Of course,
peptides and proteins which regulate transcription or
expression also recognize specific se~uence in duplexes.
In non2 of the foregoing reports, however, is there a
covalent bond ~ormed between the speciic binding agent
and the duplex.
In contrast, sequence-specific recognition of
~; single-stranded DNA accompanied by covalent crosslinking
has been reported by several groups. For example,
~ Vlassov, V.V., et al., Nucleic Acids Res (1986) 14:4065-
`~ 4076, describe covalent bonding of a single-stranded DNA
fragment with alkylating derivatives of nucleotides
complementary to target sequences. A report of similar
~ 35 work by the same group is that by Knorre, D.G., et al.,
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W091/18~97 PCT/US9l/~0
7~3
Biochimie (1985) 67:785-7~9. Iverson and Dervan also
showed sequence-specific cleavage of single-stranded DNA
mediated by incorporation of a modified nucleotide which
was capable of activating cleavage ~J Am Chem soc (1987)
109:1241-1243). Meyer, R.B., et al., J Am Chem soc
(1989~ 111:8517-8519, ef~ect covalent crosslinking to a
target nucleotide using an alkylating agent complement~ry
to the single-stranded target nucleotide sequence. A
photoactivated crosslinking to single-stranded
oligonucleotides mediated by psoralen was disclosed by
Lee, B.L., et al., Biochemistr~ (1988) 27:3197-3203.
Use of N4,N4-ethanocytosine as an alkylating
agent to crosslink to single-stranded oligonucleotid~s
has also been described by Webb and Matteurci, J Am Chem
15 Soc (1986) 108:2764-2765; Nucleic Acids Res l1986)
14:7661-7674. These papers also describe the synthesis
of oligonucleotides containing the derivatized cy~osin20
Matteucci and Webb, in a later article in Tet Lette~s
(1987) 28:2469-2472, describe the synthesis of oligomPrs
containing N6,N6-ethanoadenine and the crosslinking
properties of this residue in the context of an
oligonucleotide binding to a single-stranded DNA.
In a recent paper, Praseuth, D., et al., P~oc
Natl Acad Sci (USA) (1~88) 85:1349-1353, described
sequence-specific binding of an octathymidylate
conjugated to a photoactivatabla crosslinking agent to
both single-stranded and double-stranded DNA. A target
27-mer duplex containing a polyA tract showed binding of
;~ the octathymidylate in parallel along the polyA.
Photoactivated crosslinking of the duplex with a
p-azidophenacyl residue covalently linked to the terminus
of the octathymidylate was achieved. While sequence-
specific association occurred at the predicted region of
~- the duplex, it appeared that the crosslinking reaction
~ 35 itself was not target specific. As photoactivation was
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W~91/18~7 PCT/US91/03~0
required to form the covalent crosslink, there could be
no question of accurate sequence-specific association of
the octathymidylate to the target sequence in the 27-mer
duplex. A requirement for photoactivation, however,
seriously limits the therapeutic potential of the
crosslinking agent. Administration to a live subject
does not readily admit of this mechanism of action.
In addition, Vlassov, V.V. et al., Gene (1988)
313-322 and Fedorova, O.S. et al., FEBS (1988) 228:273-
276, describe targeting duplex DNA with a 5'-phospho-
linked oligonucleotide.
Disclosure o~ the Invention
The invention provides crosslinking agents
which associate in a sequence-specific manner to the
major groove of nucleic acid duplexes to obtain triple
helical products which are stabilized by c~valent bonds.
~he stabilized triplex may be optionally subjected to
conditions which result in cleavage of the duplex. When
applied in the context of therapeutic applications, the
stabilized binding of the sequence-specific crosslinking
;~ agent permits either interruption of the normal function
of the duplex (~or exàmple, in replication) or, in the
;~i case of regulatable duplexes (for example, associated
with transcription), may enhance the activity of the
target duplex. Depending on the nature of the covalent
bond formed as the crosslink, the resulting triple-
helical complex may become more or less susceptible to
cleavage ~nder ambient or in situ conditions.
Stimulation of cleavage may be desirable in the case of
therapeutic regimens designed to inactivate the target
DNA; it is also useful in diagnostic assays by permitting
facile detection of covalently bound probes.
In one aspect, the invention is directed to
crosslinking agents which associate with the major groove
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W~91/~g97 PCT/~91/03~80
~<~n ~ ~ 9
of nucleic acid duplexes in a sequence-speci~ic manner
and which effect at least one covalent crosslink to at
least one strand of the duplex. Multiple crosslinks may
also be for~ed, with one or both of the duplex strands,
S depending on the design of the crosslinkin~ agentO
Preferred crosslinking agents are oligonucleotides, which
take advantage of the duplex sequence-coupling rules
known in the art, and peptide sequences, which can be
designed to mimic regulatory peptides which recognize
specific sequences. The moiety which performs ~he
- crosslinXing function of the crosslinking agent results
in the formation of covalent bonds in a pattern dependent
on the design of the agent.
In an additional aspect, the invention is
directed to methods to form triple helical complexes
containing sequence-specific agents covalently bound in
the major groove, which method comprises contacting the
target duplex with a crosslinking reagent of the
invention. In still other aspects, the invention is
directed to the resulting triple helical complexes, and
to methods for therapy and diagnosis using the
crosslinking reagents of the invention.
rief Description of~he Drawings
Figure 1 shows t~e structures of preferred
alkylating agents which effect the crosslinking of the
sequence-specific agents of the invention.
Figure 2 outlines the procedur~ ~or preparation
of the N4,N4-ethanocytosine-containing oligomers that are
; 30 preferred crosslinking reagents of the invention.
Figure 3 shows the construction of a
tetracassette duplex designed to assess the specificity
of the reagents of the invention.
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W~91/18997 ~ PCT/US91/03680
Figure 4 shows the results of an assay showing
the sequence specificity of the invention crosslinXing
agent.
Figure 5 shows the results of treatment of
S target sequences with ~he reagents of the invention with
and without cleavage of the complexes~
Modes of carrvinq out the Invention
The invention provides reagents which are
capable of sequence-specific binding in the major groove
of a nucleic acid duplex and which are also capable of
- forming covalently bonded crosslinks with the strands of
the duplex without the necessity for photoacti~a~ion. As
demonstrated below, moieties to effect the covalent
bonding are employed which do not override the sequence
specificity of the remainder of the reagent. In
addition, the moiety which effects the covalently bonded
crosslink is itself specific for a particular target site
in a preferred embodiment.
sequence SpecificitY
Sequence specificity is essential to the
utility of the reagents of the invention. If not capable
of distinguishing characteristic regions of a target from
thosa of duplexes which are not to be targeted, the
reagents would not behave in a manner compatible with
their function as either therapeutic or diagnostic
agents. Accordingly, it is essential tha~ despite the
reactivity of the moiety which effects covalent binding,
~- 30 this activity not be so kinetically favored that sequence
specificity is lost.
~ Sequence specificity can be conferred in a
;~; manner consistent with the chemical nature of the
`~ reagent. In principle, the specificity is conferred by
~` 3S providing a region of spatial and charge distribution
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WO9~tl~997 P~T/U~9~/03680
~ 7~ ~
which allows close association between the reag~nt and
the charge and spatial contours of the major groove of
the target duplex. This association and sequence
specificity are defined in terms of the ability of the
reagent to distinguish between target sequences in a
sample which differ in one or more hasepairs. The
reagents o~ the invention can di~criminate between
regions of duplexes which differ by as few as 1 basepair
out of 5, preferably 1 basepair out of lO, more
preferably 1 basepair out of 15, and most preferably 1
basepair out of 20, in in vivo or in vitro c~lture
conditions or under conditions of the diagnostic assay.
The stringency of the criterion varies with the langth of
the region, since larger regions can tolerate more
lS mismatches than smaller ones under the same conditions.
Thus, a highly discriminatory reagent could detect a
mismatch of only 1 basepair in a sequence of 20
basepairs; a more sequence-specific reagent could detect
this l-basepair difference in a region of 30 basepairs.
The reagents of the in~ention are capable o~ at least
discriminating between differences of 1 basepair in a 5-
mer target, pre~erably 1 basepair in a lO-mer target, and
most preferably l basepair in a 20-mer target.
If the sequence specificity in the reagent is
conferred by an oligonucleotide, advantage can be taken
of the rules for triple helix formation in the major
groove, a~ described by Dervan (supra). These rules
continue to be developed. For classical parallel binding
of a single-stranded oligomer to a duplex, homopyrimidine
stretches bind to homopurine stretches in one strand of
the duplex wherein A associates with T and G with C,
analogous to the complementarity rules. In this mode of
a~sociation with the major groove, generally known as
parallel or CT binding, the oligomer is oriented in the
same direction, 5' ~ 3', as the homopurine stretch. An
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WO91/1~g97 PCT/US91/0368
alternate, more complex form of triple helix formation,
known as GT binding, results in an antiparallel
orientation.
Association of the oligonucleotide sequence
specificity-conferring region of the reagent can be
manipulated by utilizing either or both CT or GT binding
to one or both strands of the target duplex. In co-
pending application u.S. Serial No. 502,272, filed 29
March l990, the published counterpart of which is PCT
US90/06128, assigned to the same assignee and
incorporated herein by reference, "switchback" oligomers
are described which contain one or more regions of
inverted polarity. One application of such "switchback"
oligomers includes the ability to design reagents which
cross over between the two strands of the duplex using
parallel association with the purine regions o~ the
strands of the duplex. Alternatively, this crossover
could be effected by modifying the oligonucleotide
-~ sequence to switch back between parallel and antiparallel
~odes of association with the major groove. Thus,
sequence specificity can be designed relative to either
; or both strands of the duplex.
"Oligonucleotide" is understood to include both
DNA and RNA sequences and any other type of
polynucleotide which is an N-glycoside or C-glycoside of
a purine or pyrimidine base, or modified purine or
pyrimidine base. ~he term "nucleoside" or "nucleotide"
will similarly be generic to ribonucleosides or
ribonucleotides, deoxyribonucleosides or
deoxyribonucleotides, or to any other nucleoside which is
an N-glycoside or C-glycoside of a purine or pyrimidine
base, or modified purine or pyrimidine base. Thus, the
stereochemistry of the sugar carbons may be other than
that o~ D-ribose in certain limited residues.
WO91/189g7 P~TJus9ltO36~o
"~ucleosidel' and "nucleotide" i~clude those
moieties which contain not only the known purine and
pyrimidine bases, ~ut also heterocyclic bases which have
been modified. Such modifications include alkylated
purines or pyrimidines, acylated puri~es or pyrimidines,
or other heterocycles. "Nucleosides" or 'Inucleotides''
also include those which contain modi~ication in the
sugar moiety, for example, wherein one or more of the
hydroxyl groups are rPplaced wi~h halogen, aliphatic
groups, or functionalized as ethers, amines, and the
like. Examples of modified nucleosides or nucleotides
include, but are not limited to:
2-aminoadenosine 2~-deoxy-2-aminoadenosine
5-bromouridine 2~-deoxy-s-bromouridine
5-chlorouridine 2'-deoxy-5-chlorouridine
5-fluorouridine 2~-deoxy-5-flurouridine
5-iodouridine 2~-deoxy-s-iodouridine
5-methyluridine (2'-deoxy-5-methyluridine
is the same as thymidine)
inosine 2'-deoxy-inosine
xanthosine 2'deoxy-xanthosine
Furthermore, as the ~ anomer binds to duplexes
in a manner similar to that for the ~ anomers, one or
more nucleotides may contain this linkage.
Oligonucleotides may contain conventional
internucleotide phosphodiester linkages or may contain
modified forms such as phosphoramidate linkages. These
alternative liking groups include, but are not limited to
embodiments wherein a moiety of the formula P(O)S,
P(O)NR2 " P(O)R, P(O)OR', CO, or CNR2, wherein R is H (or
a salt)or alkyl (1-6C) and R' is alkyl (1-6C) is joined
to adjacent nucleotides through -O- or -S-. No~ all such
linkages in the same oligomer need to be identical.
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WO91/18997 PC~/U~1/036~0
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Inversions of polarity can also occur in
"derivatives~ of oligonucleotides. ~'Derivatives~ of the
oligomers include those conventionally recognized in the
art. For instance, the oligonucleotides may be
covalently linked to various moieties such as
intercalators, substances which interact specifically
with ~he minor groove of the DNA double helix and other
arbitrarily chosen conjugates, such as labels
(radioactive, fluorescent, enzyme, etc.). These
additional moieties may be derivatized through any
convenient linkage. For example, intercalators, such as
acridine can be linked through any available -OH or -SH,
e.g., at the terminal S' position of RNA or DNA, the 2'
positions of RNA, or an OH or SH engineered into the 5
position of pyrimidines, e.g., instead of the 5 methyl of
cytosine, a derivatized from which contains -CH2CH2CH2OH
or -CH2CH2C~2SH in the 5 position. A wide variety o~
substituents can be attached, including those bound
through conventional linkages.
The -OH moieties in the oligomers may be
replaced ~y phosphonate groups, protected by standard
; protecting groups, or activated to prepare additional
linkages to other nucleotides, or may be bound to the
conjugated substituent. The 5' terminal OH may be
phosphorylated; the 2'-OH or OH substituents at the 3'
terminus may also be phosphorylated. The hydro~yls may
also be derivatized to standard protecting groups.
Methods ~or synthesis of oligonuc~eotides are
found, for example, in Froehlerj B. , et al., Nucleic
Acids Research (1986) 14:5399-5467; Nucleic_Acids
Research (1988) 16:4831 4839; Nucleosides and_Nucleotides
(1987) 6:287-291. Froehler, B., Tet Lett ~1986) 27:5575-
5578; and in copending Serial No. 248,517, filed
September 23, 1988, the European counterpart of which was
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published based on EP application no. 89/3096347,
incorporated herein by reference.
In general, there are two commonly used solid
phase~based approaches to ~he synthesis of
oligonucleotides, one involving intermediate
phosphoramidites and the other involving intermediate
phosphonate linkages. In both of these, the growing
nucleotide chain is coupled to a solid support. In
conventional methods, thls linkage is as an ester formed
through a succinyl residue on the support. At the
termination of the synthesis, the oligonucleotide is
cleaved from the solid support under nucleophilic
conditions; linkage through the succinyl residue requires
reasonably strong nucleophilic conditions. The standard
conditions are concentrated ammonium hydroxide at 20C
for 2 hr.
Many of the oligonucleotides of the present
invention which are sequence-specific binding agents to
; the major groove of the double helix and provide moieties
capable o~ effecting covalent linkages, contain covalent
linking moieties which are partially destroyed by these
conditions. This disadvantage of solid-phase synthesis
is overcome according to the present invention by
` utilizing an oxalyl ester linker for coupling to the
2~ solid support. This linker is cleaved under much milder
conditions and the oligonucleotide can be released from
the support with no significant degradation of a
covalently-binding moiety such as, for example, N4,N4-
ethanocytosine. Typical condi~ions for release of t~e
oligonucleotide from the oxalyl ester are 20% aziridine
in dimethylformamide for 1 hr.
With respect to the synthesis itself, in the
phosphoramidite based synthesis, a suitably protected
nucleotide having a cyanoethylphosphoramidite at the
position to be coupled is reacted with the free hydroxyl
WO91/18997 ~Q'~ CT/U~91/~36~0
of a growing nucleotide chain deriva~ized to a solid
support. The reaction yields a cyanoethylphosphonate,
which linkage must be oxidized to the eyanoethylphospha~e
- at earh intermediate step, since the reduced ~orm is
unstable to acid. The phosphonate-based synthesis is
conducted by the reaction of a suitable protected
nucleoside containing a phosphona~e moiety at a position
to be coupled with a solid phase-derivatized nucleotide
chain having a free hydroxyl group, in the presence of a
suitable catalyst ~o obtain a phosphonate linkage, which
is stable to acid. Thus, the oxidation to the phosphate
or thiophosphate can be conducted at any point during the
synthesis of the oligonucleotide or after synthesis of
the oligonucleotide is complete. The phosphonates can
also be converted to phosphoramidate derivatives by
reaction with a primary or secondary amine in the
presence of carbon tetrachloride.
Variations in the type of internucleotide
; linkage are achieved by, for example, using the
methylphosphonates rather than the phosphonates per se,
using thiol derivatives of the nucleoside moieties and
gsnerally by methods known in the art. Non-phosphorous
based linkages may also be used, such as the formacetyl
type linkages described and claimed in co-pending
applications U.S. Serial Nos. 426,626 and 4~8,914, filed
on 24 October 1989 and ll December 1989, both assigned to
the same assignee and ~oth incorporated herein by
re~erence.
In addition to employing these very convenient
and now most commonly used, solid phase synthesis
techniques, oligonucleotides may also be synthesized
using solution phase methods such as triester synthesis.
-~ These methods are worka~le, but in general, less
efficient for oligonucleotides of any substantial length~
.
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WO 91/18997 P~/U!~;91/03680
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The parameters which a~fect the ability o~
peptide sequences to recognize particular DNA duplex
sequence targets are less well understood, but it is well
~ known that indigenous proteins are capable of regulating
i 5 transcription by selectively targeting designated regions
of the duplex. In addition, as recited in the Background
section above, specific peptides have been designed which
are capable of the desired duplex sequence recognition.
These peptides are often derivatized to additional
moieties.
The sequence specificity-conferring region of
the reagent is, thus, preferably an oligonucleotide
and/or a peptide; i.e., combinations of these modalities
may be used. However, other polymeric molecular designs
which have the appropriate spatial and charge
configuration to discriminate between duplex regions
according to the criteria set forth above, can also be
; used.
Assay fo~ Covalent Bindi~a with Tem~late
The ability of the candidate crosslinking
reagent to e~fect covalent bonding to the target duplex
can be assessed in simple assays using either a shift in
electrophoresis gel mobility or assessment of size after
cleavage. The template can be advantageously labeled at
a terminus using, for example, ~-P32 dATP and Klenow.
The labeled template and the candidate oligonucleotide
are then incubated under suitable conditions to ef fect
triplex binding. For the shift assay they are then
analyzed on a 6~ denatured polyacrylamide gel after
addition of an equal volume of formamide denaturant. The
shift in mobility verifies binding to form the triplex
and resistance to denaturation.
Reaction to form covalent linkages which then
permit cleavage to be effected is demonstrated by
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WO91/t8~7 PCT/US91/03680
~ 9
following the incubation to form tripl~x by heating with
pyrolidine at 95C for lO min to effect the cleavage.
The reaction mixture is dried down and ethanol
precipitated and analyzed on 6% polyacrylamide gel.
In both of the foregoing assays, the triplex
binding buffer depends on the temperature and pH of the
incu~ation mixture. For binding at pH 6, the incubation
is conducted at room temperature and the buffer contains
25 mM MOPS, 140 mM KCl, 10 mM NACl, 1 ~M MgC12 and 1 mM
spermine. The buffer composition is identical for pH 7.2
conditions except for the pH adjustment, and incubation
is conducted at 37OC.
In the gel mobility shift as5ay~ formation of
the triplex results in a decreased mobility; when
cleavage is effected, the size of the fragments is a
~urther indication that specific covalent linkage has
resulted in a cleavage-susceptible triplex.
A more sophisticated assay for sequence
specificity is described below.
AssaY for Sequence SPecificity
The ability of a candidate crosslinking reagent
to exhibit ~he required sequence specificity can readily
be assessed by the procedure described in detail in the
example below. ~riefly, the required elements include a
DNA duplex labeled at one terminus which contains
individual cassettes exhibiting the level of sequenc~
distinction desired. For example, each cassette might
contain a duplex of 30 bp which differs in only one
position from corresponding 30 bp stru~tures in three
other cassettes in the duplex. The candidate reagent is
reacted with the labeled test DNA containing the
cassettes, and the location of binding is determined. As
the covalent crosslinking moiety associated with the
reagent is also capable o~ effecting cleavage of the
,
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WO91/18~7 PCT/~91/036~0
-15-
duplex under appropriate conditions, the location of
binding by the reagent can readily be ascertained by
application of the sample to size separation techniques.
Multiple binding to more than one cassette will result in
multiple small fragments; binding to only one of the
cassettes results in a single defined fragment o~ the
labeled DNA of predicted size. Thus, even without prior
knowledge of design rules for specific association,
candidate reagents can conveniently be tested with
suitably labeled cassette-containing DNA.
Coval2nt~Bondinq Moietv
Included in the crosslinking aqent is a moiety
which is capable of effecting at least one covalent bond
between the crosslinking agent and the duplex. Multiple
covalent bonds can also be formed by providing a
multiplicity of such moieties. The covalent bond is
preferably to a base residue in the target strand, but
can also be made with other portions of the target,
including the saccharide or pho~phodiester. ~he reaction
nature of the moiety which effects crosslinkiny
determines the na~ure of the target in the duplex.
Preferred crosslinking moieties include acylating and
alkylating agents, and, in particular, those positioned
relative to the sequence specificity-conferring portion
so as to permit reaction with the target location in the
strand.
If the sequence specificity-conferring portion
is an oligonucleotide, the crosslinking moiety can
conveniently be placed as an ana~ogous pyrimidine or
purine residue in the sequence. The placement can be at
the 5' and/or 3' ends, the internal portions of the
seguence, or combinations of the above. Placement at the
termini to permit enhanced flexibility is preferred.
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W~91/18997 PCT/US91/0368n
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: Analogous moieties can also be attached to peptide
backbones.
: In one particularly preferred embodiment of the
crosslinking agent of the invention, a switchback
oligonucleotide containing crosslinking moieties at
either end can be used to bridge the s~rands of the
duplex with at least two covalent bonds. In addition,
nucleotide sequences of inverted polarity can ~e arranged
in tandem with a multiplicity of crosslinking moieties to
strengthen the complex.
Exemplary of alkylating moieties that are
useful in the invention are those shown in Figure l.
: These are derivatized purine and pyrimidine bases which
can be included in reagents which are oligomers of
nucleotides as described above. As seen in Figure l,
heterocyclic base analogs which provide alkyl moieties
attached to leaving groups or as aziridenyl moieties are
shown. ("Aziridenyl" refers to an ethanoamine
substituent of the formula ~N / )
It is clear that the heterocycle need not be a
purine or pyrimidine; indeed the pseudo-base to which the
reactive function is attached need not be a heterocycle
` at all. Any means of attaching the reactive group is
satisfactory so long as the positioning is correct.
Additio~al Com~onents of the Crosslinkinq A~ents
While the crosslinking agents of the invention
- re~uire a sequence specificity conferring portion and a
moiety which effects covalent crosslinking to the duplex,
the agent can also contain additional components which
provide additional functions. For example, ligands which
effect transport across cell membranes, specific
targeting of particular cells, stabilization of the
triplex by intercalation, or moieties which provide means
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W~91/~997 PCT/~S91~03680
--17--
for detecting the oligomer alone or in the context of the
triple helix formed can be included. The crosslinking
: ag2nts of the invention may thus be further conjugated to
lipid-soluble components, carrier particles, radioacti~e
or fluorescent labels, specific targeting agents ~uch as
antibodies, and mem~rane penetrating agents and the like.
Utility and Administration
The specific crosslinking agents of the
invention are useful in therapy and diagnosis. In
general, in therapeutic applications, the agents are
designed to target duplexes for either interruption or
enhancement of their function. For example, suitable
target genes for enhanced function include thsse which
control the expression of tumor suppressor genes ~Sager,
Science (1989) 246:1406) or for duplexes which control
the expression of cytokines such as IL-2. By redesign of
the oligomer, however, complexing into the major groove
may result in blocking the func~ion of the target duplex
as would be desirable where the duplex mediates the
progress of a disease, such as human immunodeficiency
virus, hepatitis-B, respiratory syncytial virus, herpP.s
simplex virus, cytomegalovirus, rhinovirus and influenza
virus. In addition, other undesirable duplexes are
formed in various malignancies, including leukemias,
lung, breast and colon cancers, and in other metabolic
disorders.
The formulation of the crosslinking agents of
the invention depends, of course, on their chemical
nature, and on the nature of the condition being treated.
Suitable formulations are available to those of ordinary
skill, and can be found, for example, in Reminqton's
Pharmaaeutical Sciences, latest edition, Mack Publishing
Co., Easton, PA. Dosage levels are also determined by
the parameters of the particular situation, and as is
:
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WO91/1$~7 PCT/US91/03680
o~r~S7~3
-18-
ordinarily required in therapeutic protocols,
~: optimization of dosage levels and modes of admini~tration
are within ordinary and routine experimentation~
The crosslinking agents of the invention are
5 particualrly useful in the treatment of latent infections
: such as HIV or HSV. For diagnostic use, protocol~ are
employed which depend for their specificlty on the
: ability of the crosslinking agent stably to bind a target
double-helix region, and which permit the detection of
this binding. A variety of protocols is available
including those wherein the crosslinking agent is labeled
to permit detection of its presence in the complex.
The following examples are intended to
illustrate but not to limit the invention.
Exam~le 1
Se~ue~L~ LI~ic Binding of Oliqomers
Containinq N4N4Ethanoc~tosine
Two 19-mers, Az-A:
5 TCTCXCTCTCTTTTTCCTT3
and A2-~:
5' 3'
TCTCTCTCTXTTTTTCCTT
. 4 4
whereln X represents N N -ethanocytosine deoxynucleotide
are synthesized as outlined in Figure 2. The steps in
the synthesis refer to Webb and Matteucci, Nucleic Acids
30 Res ~1986) 14:5399-5467 and Froehler and Matteucci,
:. Nucleic Acids Res (1986) 14:7661_7674; the sec~nd step is
also described in Marugg et al., Tet Lett (198_) 27:2661.
~' The 19-mers were recovered and puri~ied using standard
~, procedures.
: 35
.~:
~O91/1~997 PCT/VS91/03680
7 ~9
--19--
.
Az-A and Az-B were tested for their ability to
bind to a labeled diagnostic DNA containing 4 test
cassettes which is diagramed in Flgure 3.
As shown in Figure 3, the test cass~ttes
contain identical sequences excep~ for a sinyle base.
Az-A is designed to associate specifically with cassette
l; Az-B is designed to associate specifically with
cassette 2. This target DNA is an end-la~eled PvuII-Sal
fragment containing these cassettes separated by
convenient restriction sites. The N4N4 cytosine moiety
was expected to crosslink covalently only to a guanine
residue.
Four identical reactions were set up: Reaction
mix 1 contained the target DNA treated with DMS which is
known to effect random covalent bonding and result in
multiple cleavage sites in the cassette. Reaction mix 2
contained Az-A at 50 ~M; reac~ion mix 3 contained Az-~ at
50 ~M. Reaction mix 4 was another control which
contained no reagent.
All reaction mixtures were a total of 10 ~l and
contained 1 ~l 10 x buffer, which contains l M NaCl, 0.2
MES, 0.1 M MgCl2, pH 6Ø The target plasmid was
supplied in 1 ~l volume at 50,000 cpmt~l, Az-A and Az-B
were supplied in 1 ~l aliquots of 500 ~M concentration
and the volume was made up in all reaction mixtures to 10
~1 with water.
The mixtures were incubated for 13.5 hr at room
temperature (23-25C).
After incubation, l ~l DMS (1.25 dilution in
:~ 30 H2O) was added to reaction mix 1 and incubated for 2 min
: - at 25C. Then all reaction mixtures received lO ~l of 2
:` M freshly diluted pyrrolidine to effect cleavage at
:~ covalent binding sites and then were further incubated
~or 15 min at 95C, placed on ice for 5 min and dried
under vacuum.
.. . . . .
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WO91/l8~7 PCT/US91/036B0
-20-
The samples were resuspended in 25 ~l water and
dried under vacuum twice and then resuspended in 6 ~l 67%
formamide, heated for 3 min at 95C and loaded onto a 6%
denaturing polyacrylamide gel. The results of denaturing
PAGE on these mixtures is shown in Figure 4.
Lane 1 represents reaction mix l to which DMS
was added. Extensive degradation is seen. Lane 2 is the
reaction mixture which contained Az-A. As shown,
treatment with pyrrolidine yields mainly one degradation
product, the size of which corresponds to the labeled
fragment that would be obtained if cleavage occurred in
cassette 1. Lane 3 shows the results from reaction mix 3
containing Az-B. Again, a single prominent degradation
fragment was obtained which corresponds in size to the
15 labeled fragment which would be ob~ained i~ cleavage
occurred in cassette 2. The pyrrolidine control in lane
4 shows only modest random degradation.
As seen from a comparissn of the sequences of
Az-A and Az-B, each specifically recognizes the
appropriate cassette differing only in one nucleotide of
19. Both also specifically covalently bind to guanine.
Example 2
SYnthesis of Oliaonucleotides 2-6
; 25 Several o~ the oligonucleotides, 2-6, as shown
in Table 1, include the base analogs aziridinylcy~osine
(N4,N4-ethanocytosine), designated "Z" in the tabulated
sequences and 5-methylcytosine, designated C' in the
; table. In the table, X indicates 1,3-propanediol.
Table 1
(2) Control 5'-C'TTTTTTTC'TTTTTC' TTC ' X
~ 3 ) 5 1 5 ' - Z TTTTTTTC ' TTTTTC ' TTX
;~ 35 (4) 3' 5'-TTTTTTTC'TTTTTC'TTZX
`~091/18997 PCTtUS91/03680
2~ 3
--21--
(5) 51 + 3, 5'-Z TTTTTTTC'TTTTTC'TTZX
t6) Internal 5'-TTTTTTTZ TTTTTC'TTX
In thP oligomer synthesis, the 5-methyl-C groups were
FMOC-protected and an oxalyl-CPG support (R. Letsinger,
personal communication, described below) wa~ used for the
synthesis.
The synthesis scheme for aziridinylcytosine is
as described in Example l. It is incorporated into the
oligomers using the standard solid phase technology
modified as follows.
The base representing the 5' terminus was
coupled to a CPG support for the production of the ODNs
using the following method (R. Letsinger, personal
communication). Oxalyl chloride (20 ~l, 0.23 mmol) was
added to a solution of 1,2,4-triazole (77 mg, l.l mmol)
in acetonitrile (2 ml). A small amount of precipitate
formed but disappeared after addition of pyridine (0.1
ml). The nucleoside at the 5' terminus (0.23 mmol) in
acetonitrile (l ml) and pyridine (0.5 ml) was added, and
after one hour the solution was drawn into a syringe
containing aminopropylsilyl-controlled-poreglass (CPG)
(400mg; 80-100 mesh, 500 A pore). This mixture was
allowed to stand for 15 min. and the liquid was ejected
and the solid washed four times with acetonitrile. Any
residual amino groups were capped by drawing in equal
volumes of THF solutions of DMAP (O.3 M) and acetic
anhydride (0.6 M). The support was then washed with
pyridine and acetonitrile and dried.
After the oligomers were synthesized, the
support bound H-phosphonate oligomer was oxidized with
I2/pyridine/H20 twice for 30 min and subsequently
converted to the free oligonucleotide by deprotection and
cleavage from the support by treatment with 20~ aziridine
in DMF for 2 hours at room temperature. The oligomers
WO 91/189g7 ,~ 3 PCT/U~91/0368Q
were recovered and further purified by running the
reaction mixture from the synthesis machine over NAP-5
(Pharmacia Sephadex G-25) column to remove salts, free
aziridinylcytosine residues, FMOC blockers, etc. The
NAP-5 column was used according to the manuracturers
directions.
Exam~le 3
AssaY for Crosslinked Triple Helix
Oligodeoxyribonucleotides 2-6 were designed to
bind the duplex target of the sequence:
5'-CCATGGAlO¦GAAAAAAAGAAAAAGAAG¦AAATTTCT~TTTCTTTCT12.. p
As a comparison.of the squared portion of the duplex to
the sequences in Figure 1 will demonstrate, the
potentially covalent binding moiety, Z, is at the 3'
terminus of the oligomer in ODN3, at the 3' end in ODN4,
at both ends in ODN5 and internal to the oligomer in
ODN6.
Each of these oligomers were incubated with the
duplex using the triplex binding buffer as set forth
above at pH 7.2 at 37C for 2 hr. The reactions were
quenched with pyrolidine, heated and evaporated as
; 25 described above before subjecting the mixtures to
: ~ denaturing PAGE. The treatment results in cleavage of
the duplex at the site of covalent bonding as described
by Maxam, A. et al., Proc Natl Acad Sci USA (1977)
. 74:560.
. 30 The results are shown in Figure 5. In Figure
: 5, lane 1 represents the untreated duplex target, and
shows no difference from lane 2 which was treated with
:~ ODN2, containing no crosslinking moiety. Lanes 3 and 4
represent the results of reaction mixtures using ODNs 3
and 4 respectively; in both cases, considerable reaction
: . - ~: . .
~091~18~7 PCT/~S91/036~0
.Lg
has occurred; this reaction is virtually complete in lane
5 which represents treatment with ODN5. Lane 6 indicates
that although some reactio~ occurred with ODN6, this wa~
less effective when the covalent binding moiety is
internal to the oligomer.
Lanes 7-10 represent the alternate form of the
assay described hereinabove wherein a mobility shift is
d~tected, rather than cleavage. In the samples applied
to these lanes, the reaction was stopped not with
pyrolidine but with ~he denaturing agent formamide.
Lane 7 represents the target duplex only, lane 8 the
target with ODN2 containing no cov~lently-binding moiety,
and lanes 9 and 10 contain reaction mixtures of the
duplex with ODNs 3 and 4 respectively. As shown in
Figure 5, the lower mobility is reflected in cases where
the covalent bonding is effected. Denaturation with the
~ormamide destroys the triplex when no crosslinking
moiety is present.
In addition, the foregoing techniques were used
to assess the kinetics of the crosslinking reaction. ~he
half-life of the reaction was approximately 1 hr for ODN4
with the concentration of ODN4 at 1 ~M; ODN3 which has
the analog at the 5' position showed a rate approximately
` four times slower. ODN4 provided virtually 100%
` 25 crosslinking after 16 hr.
-~ Example 4
Additiional Crosslinking Aaents
In the illustrative oligonucleotides set forth
below, the ~ollowing notation is used: The modified
nucleoside N-methyl-8-oxo-2'-deoxyadenine (MODA) is
designated "M"; 5-methylcytosine is represented by "C";
and nucleosides containing an aziridenyl group (N4N4-
ethanocytosine) are designated "Z".
.: .: - - . .
,,
.: : - . .
WO91/18~7 ~ PCT/US91/03680
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In addition, some of the oligomers contain an
inverted polarity region, in this illustration formed
from an o-xyloso dimer synthon. The linking group, o-
xyloso (nucleotides that have xylose sugar linked via the
o-xylene ring), is designated "X".
Crosslinking agents that bind to certain HIV
targets are as follows. For binding ~o the 5~-
GGAAAAGGAAGGAAATTTC-3' sequence:
111 5l-MMTTTTMMTTMMT-X1-TTM-5';
112 5'-MMTTTTMMTTMMT-Xl-TTZ-5';
113 5'-ZMTTTTMMTTMMT-Xl-TTZ-5';
114 5'-ZMTTTTMMTTMMT-Xl-TTM-5';
115 5'-MCTTTTMCTTMCT-Xl-TTM-5';
116 5'-MCTTTTMCTTMCT-Xl-TTZ-5';
117 5'-ZCTTTTMCTTMCT-Xl-TTZ-5'; and
118 5'-ZCTTTTMCTTMCT-Xl-TTM-5'.
For binding to the 5'-AGAGAGAAAAAAGAG-3'
~equence:
: 131 5'-TCTCTCTTTTTTCTC-3';
132 5'-TCTCTCTTTTTTCTZ-3';
133 5'-ZTCTCTTTTTTCTZ-3'; and
134 5'-MTMTMTTTTTTMTZ-3'.
For binding to the 5'-AAGAGGAGGAGGAGG-3'
sequence:
141 5'-TTCTMCTMCTMCTMZ-3';
142 5'-TTCTMMTMMTMMTMZ-3'; and
143 5'-TTCTCMTCMTCMTCZ-3'.
For binding to the S'-AGAAGAGAAGGCTTTC-3'
sequence:
. 30 152 5'-TCTTCTCTTM-X2-TTZ-5'; and
156 5'-TMTTMTMTTM-X2-TTZ-5'.
The oligonucleotides are labeled by kinasing at
the 5' end and are tested for their ability to bind
target sequence under conditions of 1 mM spermine, 1 mM
MgC12, 140 mM XCl, 10 mM NaCl, 20 mM MOPS, pH 7.2 with a
.. : . .. .
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, ~ .. .
WO91t18997 PCT/US9~/03680
~ $~ ~
target duplex concentration of lO pM at 37C for 1 hour.
These conditions approximate physiological conditions,
and the binding is ~es~ed either in a footprint assay, or
in a gel-shift assay essentially as described in Cooney,
M. et al., Science (1~88) 241:456-4590
For oligomers designed to target ~uman
Interleukin-1 Beta Gene (HUMILlB), illustrative
nucleotides are ;
a. for HUMILlB beginning at neucleotide 6379
104 5'-ZTTTTMTTMTM-X1-TMTTTT-5',
b. for HuMILls beginning at neucl~otide 7378
112 5'-ZTTCTTTTTTTTT-X2-CTTTCMT-5',
114 5'-MTTMTTTTTTTTT-X2-MTTTMZ-5',
115 5'-ZTTMTTTTTTTTT-X2-MTTTMZ-5l,
116 5'-ZTTMTTTTTTTTT-X2-MTTTMM-5l.
For oligomers designed to target Human Tu~o~
Necrosis Factor (HUMTNFAA), the illustrative nucleotides
are:
a. for HUMTNFAA beginning at neucleotide 251
203 5'-TMTMMMTTM-X3-MMMMZ-5',
: b. ~or HUMTNFAA beginning at neucleotide 1137
212 5'-ZMMMTTCTCTCTCTCTCTTTCT-3',
214 5'-MMMMTTCTCTCTCTCTCTTTZ-3',
:: 215 5'-ZMMMTTCTCTCTCTCTCTTTZ-3',
216 5'-ZMMMTTCTCTCTCTCTCTTTM-3',
218 5'-MMMMTTMTMTM~MTMTMTTTZ-3',
219 5'-2MMMTTMTMTMTMTMTMTTTZ-3',
220 5'-ZMMMTTMTMTMTMTMTMTTTM-3'.
For oligomers designed to target Human
Leukocyte Adhesion Protein pl50,95 Alpha Subunit Gene
(HUMINT02), illustrative nucleotides are:
a. for HUMINTO2 beginning at neucleotide 1612
302 5'-TCTTMCTT-X4-MTTCTMZ-5',
304 5'-TMTTMMTT-X4-MTTMTMZ 5',
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Wo 91/i8997 PCr/US91/03680
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26-
For oligomers designed to target Human
Interleukin-2 Receptor Gene (HUMIL2R8), the exon 8 target
- and flanks, illustrative nucleotides are:
a. for HUMIL2R beginning at neucleotide 1114
502 5'-TTMCTTMCTTTCTTTCTTMCTTZ-3',
504 5'-MM~TMMTTTMTTTMTTMMTTZ-3',
505 51-ZMTTMMTTTMTTTMTTMMTTM-3',
506 5'-ZMTTMMTTTMTTTMTTMMTTZ-3',
b. for HUMIL2R8 beginning at neu~leotide 1136
512 5'-ZTTCTMMMTCTTMMMT-3'.
For oligomers designed to target Human
Interleukin-4 Gene (HUMIL4), the illustrative nucleotides
are:
a. for HUMIL4 beginning at neucleotide 75
602 5 ' -TNTMMMNNTTZ-3 ',
b. for HUMIL4 beginning at neucleotide 2~6
612 5'-ZTCTTMMT-X6-MTTMT-3',
614 5'-ZTMTTMMT-X6-MTTMT-3'.
For oligomers designed to target Human
Interleukin-6 Receptor Gene (HUMIL6), the illustrative
nucleotides are:
, a. for HUMIL6 beginning at neucleotide 2389
702 5'-ZMMMTTCT-X6-TMTMTMMTMMMTTTMTTMMT-5',
704 5'-MMMMTTCT-X6-TCTCTCCTMMMTTTMTTMNZ-5',
. 25 705 5'-ZMMMTTCT-X6-TCTCTCCTMMMTTTMTTMMZ-5',
706 5'-ZMMMTTCT-X6-TCTCTCCTMMMTTTMTTMMM-5',
b. ~or HUMIL6 beginning at neucleotide 2598
712 5' TMTMMTTMMTMTMMTNTMMMZ-3',
714 5'-TMTMCTTMCTMTMC~MTMMMZ-3'.
For oligomers designed to targat Human
Interleukin-6 Gene ~HUMIL6B), the sequence beginning at
neucleotide 18, the illustrative nucleotides are:
:, 802 5'-ZTMMMMTTMTM-Xl-TTMT-5'.
:~,
:'
~;
... . . . . .
.
W~9l/18997 PCTt~91/~3680
2 ~ ~IL~
For oligomers designed to target Human
Interferon-Gamma Gene (HUMINTGA), the sequence beginning
at neucleotide 295, the i-llustrative nucleotides are:
812 5'-MMTTTMTMMTMTZ-3',
813 5l-ZMTTTMTMMTMT2-3~,
814 S;-ZMTTTMTMMTMTM-3'.
For oligomers designed to target Human
Interleukin-l Receptor Gene (HUMILlRA), the illustrative
nucleotides are:
a. for HUMILlRA beginnin~ at neucleotide 3114
912 5'-TTTMMTMMTMMTT~Z-3',
914 5'-TTTMCTMCTMCTTMMZ-3'.
For oligomers designed to target Human Tumor
Necrosis Factor Receptor mRNA (XUMNFR), the se~uence
beginning at nucleotide 2354:
942 5'-TTTTCTTTTTTTTTTTTZ-3',
943 5'- TTTTMTTTTTTTTTTTTZ- 3~.
For oligomers designed to targQt Human
Hepatitis B Virus (HBV), the illustrative nucleotid~s
are:
a. for HBV beginning at nucleotide 2365
101 5'-TCTTCTTCT-X1-~MMT~ 5',
102 5'-TCTTCTTCT-X1-MMMTZ-5',
103 5'-TMTTMTTMT-Xl-MMMTM-5',
104 5'-TMTTMTTMT-Xl-MMMTZ-5',
b. for HBV beginning at nucleotide 2605
111 5'-MTCTTTTCTTCT-3',
112 5'-ZTCTTTTCTTCT-3',
113 5'-MTMTTTTMTTMT-3',
114 5'-ZTMTTTTMTTMT-3'.
For oligomers designed to target Human
Papilloma Virus Type 11 (HPV-ll), the illustratiYe
`: nucleotides are:
a. for HPV-ll beginning at nucleotide 927
201 5'-MTMCTTCTMCTMC-3',
--
. . .. . . .. .
WO91/18997 PCT/US91/03680
19
-28-
202 5'-ZTMCTTCTMCTMC-3',
b. for HPV-11 beginning at nucleo~ide 7101
211 5'-TTTTCTTT-X1-TTTM-5',
2 12 5 ' -TTTTCTTT - X 1 -TTT Z--5 ',
213 5'-TTTTMTTT-X1-TTTM-5',
214 5l-TTTTMTTT-Xl-TTTZ-5l.
For oligomers designed to target Human
Papilloma Virus Type 16 ~HPV-16), the sequence beginning
at nucleotid~ 6979, the illustrative nucleotides are:
301 5'-TTTMCTTT-X1~TTCT-5',
302 5'-TTTMMTTT-Xl-TTMT-5'.
For oligomers designed to target Human
Respiratory Syncytial Virus (RSV), the illus~rative
nucleotides are:
a. for RSV beginning at nucleotide 1307
401 5'-TMCTTCTCTTCT-3',
402 5'-TMMTTMTMTTMT-3',
403 5'-TCCTTMTMTTMT-3',
b. for RSV beginning at nucleotide 5994
411 5'-TTCTTTTMCTTTTCT-X1-TTCTT-5',
412 5'-TTMTTTTMMTTTTMT-X1-TTMTT-5'.
For oligomers designed to target Herpes Simplex
Virus II tHSV II IE3), the illustrative nucleotides are:
501 5'-MTCTTCTTCTT-X2-MCMCMCMCM-5',
502 5'-MTCTTCTTCTT-X2-MCMCMCMCZ-5',
. 503 5'-ZTCTTCTTCTT-X2-MCMCMCMCZ-5',
504 5'-ZTCTTCTTCTT-X2-MCMCMCMCM-5',
505 5'-MTCTTCTTCTT-X2-~MMMMMU~-5l,
506 5'-MTCTTCTTCTT-X2-NM~H~Z-5',
507 5'-ZTCTTCTTCTT-X2-N~WM~MZ-5',
508 5'-ZTCTTCTTCTT-X2-NMNM~MM~-5',
509 5'-MTMTTMTTMTT-X2-M~MNM~M-5',
. ~ 510 5l-MTMTTMTTMTT-X2-MM~MMMMMZ-5l,
511 5'-ZTMTTMTTMTT-X2-M~MMM~MMZ-5',
512 5'-ZTMTTMTTMTT-X2-MMM~M~M~M-5'.
,
;
W~91/18997 PCT/U~91/~368
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-29-
For oligomers designed to target Herpes Si~plex
Virus II (HSV II Ribonucleotide Reductase), the
illustrative nucleotides are:
601 5'-MTMMMMMM-X3-CTTCTTM-5',
602 5'-MTM~MM~M-X3-CTTCTTZ-s',
603 5'-ZTMMMMMM-X3-CTTCTTZ-5',
604 5'-ZTM~MMMM-X3-CTTCTTM-5',
605 5'-MTMMMMMC- X3-MTTMTTM- 5',
606 5'-MTMMMMMC- X3-MTTMTTZ-5',
607 5'-ZTMMMMMC-X3-MTTMTTZ-5',
608 5'-ZTMMMMMC-X3-MTTMTTM-5'.
For oligomers designed to target Herpes Simplex
Virus I (HSV), the illustrative nucleotides are:
a. for HSV beginning at nucleotide 5~916
: 15 701 5~-MMMTTTMCTTTMTMCTTT-3',
702 5'-MMMTTTMMTTTMT~MTTT-3~,
703 5'-MMMTTTCCTTTMTCCTTT-3l,
b. ~or-HSV beginning at nucleotide 121377
: 711 5' MTMMMTM-X3-TMCTCTT-5',
;~ 20 712 5'-ZTMMMTM-X3-TMCTCTT-5',
713 s'-MTMMMTM-X3-TMMTMTT-5',
714 5'-ZTMMMTM-X3-TMMTMTT-5',
c. for HSV beginning at nucleotide 10996
.~ 721 5'-MMMMMTCTMMM-X1-TMMMTCT-5',
722 5'-2MMMMTCTMMM-Xl-TMMMTCT-5l,
723 5'-MMMMMTMTMMM-Xl-TMMMTMT-5',
-~ 724 5'-ZMMMMT.MTMMM-X1-TMMMTMT-5'.
. For oligomers designed to target
Cytomegalovirus (CMV), the illustrative nucleotides are:
~ 30 a. for CMV beginning at nucle~tide 176
: 801 5'-MMMMTTTTMTMMT-X1-TMMM-5',
802 5'-MMMMTTTTMTMCT-Xl-TMMM-5',
803 5'-MMMMTTTTMTMCT-X1-TMMZ-5',
804 5'-ZMMMTTTTMTMCT-Xl-TMMZ-5',
805 5'-ZMMMTTTTMTMCT-X1-TMMM-5',
::;
WO 91tl8997~q~$~ 3 PCI/US91/03680
--30--
b. ~or CMV beginnlng at nucleotide 37793
811 5'-MMMTTCTM-X3-CTTCTMMMM-5',
812 5'-MMMTTCTM-X3~CTTCTMMMZ 5',
813 5'-ZM~TTCTM-X3-CTTCTMMMZ-5',
814 5'-ZMMTTCTM-X3-CTTCTM~M-5',
815 5'-MMCTTMTM-X3-MTTMT~MMM-5',
816 5'-MMCTTMTM-X3-MTTMTMM~Z-5',
817 5'-ZMCTTMTM-X3-MTTMTMMMZ-5',
818 5'-ZMCTTMTM-X3-MTTMTMMMM-5',
c. for CMV beginning at nucleotide 7304
821 5'-MMMMTMCTCTMCTCTCTCTTCTMCTM-3',
822 5'-M~ TMCTCTMCTCTCTCTTCTMCTZ-3',
-~ 823 5'-MMMMTMMTMTMMTMTMTMTTMTMMTM-3~,
~ 824 5'-MMMMTMMTMTMMTMTMTMTTMT~TZ-3',
`. 15825 5'-ZMMMTMMTMTMMTMTMTMTT~MMTZ-3l,
826 5'-ZMMMTMMTMTMNTMTMTMTTMTMMTM-3',
:, 827 5'- ~ TCCTMTCCTMTMTMTTMTCCTM-3',
828 5'-MMMMTCCTMTCCTMTMTMTq~'CCTZ-3',
829 5'-ZMMMTC~CTMTCCTMTMTMTTMTCCTZ-3',
830 5'-ZMMMTCCTMTCCTMTMTMTTMTCCTN-3'.
:
~:.
............ . .
- . ~. . ~ . -
.. . .
, . ~ ~ , .
- ~ , .
