CA2083719A1 - "sequence-specific non-photoactivated crosslinking agents which bind to the major groove of duplex dna - Google Patents

Abstract

Agents which bind to the major groove of nucleic acid duplexes in a sequence-specific manner and are capable of forming covalent bonds with one or both strands of the duplex in the absence of light are useful therapeutic agents in the treatment of conditions mediated by duplex DNA. These agents are designed so that the reactivity of the crosslinking agent does not interfere with the sequence specificity of the agent which binds to the major groove.
Thus, specific desired DNA duplexes can be targeted and their activity diminished or enhanced.

Description

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 , : , ., j. ~ .

~ WO91/189s7 PCT/US9~tO36X0 i~ $~1~3 ~ 2-:
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., ; ::

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 `: :
. . .

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 ~'` ' :
~ .

. ~ , , 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.

' ,,. , , ,, ,, ~ , ,' .
~' ' '' , 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 ,~

, , . .
:. . " , . . : . .
-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 ~' ' .

, ~r ~r~
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.

-.. ~, -:' WO91/18997 PC~/U~1/036~0 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 ~ ' ' .
- : . , ` WO 91/lX~7 ~ 7~ PCT/V~9~/03680 . - .

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 ~$~

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 ' ' , "~ ' ' !
~' .

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 , ' WO91/18~7 PCT/~91/036~0 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.

;

. - -W~91/18997 PCT/US91/0368n ~ ~ ~53 ;' : 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 ' ' , : ~.

W~91/~997 PCT/~S91~03680 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 :

. : .

WO91/1$~7 PCT/US91/03680 o~r~S7~3 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:

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 .
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 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 (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 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 .. : . .. .

.. , -, ~ .. .

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', , ~ , . . .

, Wo 91/i8997 PCr/US91/03680 ~ LJ

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', --. . .. . . .. .

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 ~9 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 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'.

:
~:.

............ . .

- . ~. . ~ . -.. . .
, . ~ ~ , .
- ~ , .

Claims (15)

Claims

1. A peptide or oligonucleotide crosslinking agent that binds in the major groove of a nucleic acid duplex in a sequence-specific manner, and which agent forms, without photoactivation, a covalent crosslink at at least one site of said duplex, said agent comprising a region conforming sequence-specificity and a moiety which effects a covalent crosslink through a residue of the peptide or a base of the oligonucleotide.

2. The crosslinking agent of claim 1 wherein the sequence specificity conferring region is an oligonucleotide or derivative thereof.

3. The crosslinking agent of claim 1 which comprises a multiplicity of moieties which effect crosslinks to the duplex.

4. The crosslinking agent of claim 1 wherein said moiety which effects crosslinking is an alkylating agent.

5. The crosslinking agent of claim 4 wherein said alkylating agent is an ethanoamino moiety.

6. The crosslinking agent of claim 5 wherein said alkylating agent is an N,N-ethanopurine or N,N-ethanopyrimidine.

7. The crosslinking agent of claim 1 wherein the moiety which effects crosslinking is a substituent of the agent selected from the group consisting of formulas 1-4 of Figure 1.

8. The crosslinking agent of claim 1 wherein said sequence specificity region distinguishes regions of the target duplex which differ by 1 bp in a sequence of 5 bp.

9. A triple helical complex which comprises a nucleic acid duplex containing the crosslinking agent of claim 1 in its major groove.

10. A method to form a covalently bonded triple helical complex with a sequence-specific agent crosslinked in the major groove, which method comprises contacting a nucleic acid duplex with the crosslinking agent of claim 1 under conditions which favor formation of said complex.

11. A method to control diseases or conditions in an animal subject, which diseases or conditions are mediated by nucleic acid duplex, which method comprises administering to a subject in need of such treatment an effective amount of the crosslinking agent of claim 1.

12. The method of claim 11 wherein said disease or condition is a latent infection.

13. A method to detect a nucleic acid duplex containing a target sequence of nucleotides, which method comprises:
contacting a sample suspected to contain said duplex with a crosslinking agent capable of covalently binding to the major groove of the duplex in a manner specific to said target sequence under conditions wherein said duplex and crosslinking reagent form a complex, and detecting the formation of at least one crosslink in said complex.

14. The method of claim 13 wherein said detecting comprises treating said complex with a denaturing agent and subjecting the resultant to denaturing electrophoresis, and wherein complexes containing said crosslink exhibit lowered mobility.

15. A method to synthesize an oligonucleotide containing at least one nucleotide residue having an ethanoamino moiety as a substituent on the base portion thereof which method comprises conducting solid-phase synthesis of said oligomer in a solid-phase system wherein the oligomer intermediates are coupled to the solid phase through an oxalyl moiety.