WO1993018187A1 - Triple helix recognition of dna - Google Patents

Triple helix recognition of dna Download PDF

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Publication number
WO1993018187A1
WO1993018187A1 PCT/US1993/002352 US9302352W WO9318187A1 WO 1993018187 A1 WO1993018187 A1 WO 1993018187A1 US 9302352 W US9302352 W US 9302352W WO 9318187 A1 WO9318187 A1 WO 9318187A1
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oligonucleotide
target sequence
purine
nucleotide
moiety
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PCT/US1993/002352
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French (fr)
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Peter B. Dervan
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California Institute Of Technology
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Priority to US07/850,503 priority
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16Purine radicals
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6839Triple helix formation or other higher order conformations in hybridisation assays

Abstract

Oligonucleotides and processes for their use for specific recognition of a target sequence in double-stranded nucleix acid through the formation of a triple helix. The oligonucleotides contain nebularine and bind to one of the strands of the target sequence. The oligonucleotides can be used as diagnostic or therapeutic agents through incorporation of an appropriate moiety in one or more nucleotides in the triple helix forming oligonucleotide.

Description

TRIPLE HELIX RECOGNITION OF DNA

FIELD OF THE INVENTION

This invention relates to the use of oligonucleotide probes containing one or more nebularine nucleotides for sequence-specific recognition of double-helical nucleic acids through formation of triple helices.

BACKGROUND OF THE INVENTION

The ability of homopyrimidine oligonucleotides to bind to homopyrimidine-homopurine tracts within large double-stranded DNA to form a triple helix was first described by Moser and Dervan (1987) Science 238:645- 650. Such oligonucleotides bind in the major groove in a parallel orientation to the homopurine strand. The isomorphous based triplets formed comprised T*AT and C«GC. Since then numerous publications have appeared relating to triple helix formation including the description of a second structural motif for triple helix formation wherein a purine-rich oligonucleotide binds in the major groove of a double-helical DNA in an anti-parallel orientation to the purine strand. Beal, P. A. , and Dervan, P. B. (1991) Science 251:1360-1363. The proposed base triplets for such triple helix formation are G«GC, AΑT and TΑT.

Notwithstanding the foregoing, the specific recognition of the the other base pairs CG and TA would provide improved targeting of single sites in large size double-stranded DNA.

SUMMARY OF THE INVENTION

In one aspect, the invention provides triple helices comprising a large double-helical nucleic acid and an oligonucleotide containing the nucleotide nebularine bound to a target sequence within the double-helical nucleic acid. In specific embodiments, the target sequence comprises a purine-rich sequence on one of the strands of the double-helical nucleic acid which contains at least one or more of the pyrimidine nucleotide C. The triple helix formed comprises an oligonucleotide containing a nebularine nucleotide when the nucleotide at the complementary position in the purine-rich target sequence contains C.

The invention also includes synthetic triple helix forming oligonucleotides capable of binding to a purine-rich target sequence containing one or more C nucleotides in a large double-helical nucleic acid. Such triple helix forming oligonucleotides contain nebularine when the complementary position in the purine-rich target sequence is C. Such oligonucleotides also preferably contain a G when the nucleotide in the complementary position of the purine-rich target sequence is G and a T or A when the nucleotide in the complementary position in the purine-rich target sequence is an A and the nucleotide

The oligonucleotide of the triple helices and the synthetic triple helix forming oligonucleotide can optionally contain in addition a nucleotide to which is attached at least one moiety. Such a moiety can be a detection moiety so as to permit detection of triple helix formation, a cleaving moiety capable of cleaving the double-helical nucleic acid to localize the site of triple helix formation or a therapeutic agent wherein triple helix formation targets the action of the therapeutic agent.

In addition, the invention includes processes for forming the above triple helices wherein a nebularine containing oligonucleotide is contacted with a large double-helical nucleic acid to form a triple helix.

The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate some of the embodiments of the invention and, together with the description, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the models for base triplets N-AT, N-CG, N*GC, and N*TA formed between 2'- deoxynebularine and the Watson-Crick duplex within the pur»pur»pyr triple-helix motif. All bases are depicted with anti glycosidic bonds and the phosphate-deoxyribose backbone of the third strand was positioned as to be compatible with the purine triple-helix motif.

Figure 2 depicts the sequences of oligodeoxy- nucleotide-EDTA 1-5 (SEQ ID NO:l) wherein T* indicates the position of thymidine-EDTA. The oligodeoxynucleotides differ at one base position indicated in bold type to the four common natural DNA bases (A, G, C, T) and to 2•-deoxynebularine (N) . Also shown are the sequences of the target double- helical DNA (SEQ ID NOS:2 and 3). The box indicates the double-stranded sequence bound by oligodeoxynucleotide-EDTA*Fe(II) 1-5 (SEQ ID NO:l). The Watson-Crick base pair (AT, GC, CG, or TA) opposite the variant base in the oligodeoxynucleotide are in bold type. The height of the arrows represent the relative cleavage efficiencies at the indicated bases as determined by quantitative analysis using storage phosphor autoradiography.

Figure 3 is a histogram depicting relative cleavage intensities (normalized) for the twenty base triplets. The values were obtained by phosphorimager quantitative analysis and represent the mean +/- standard diviation of two determinations.

Figure 4 is a ribbon model and sequence of the triple helix complex between a single site in the pULHIV Eco01091-Sspl restriction fragment (SEQ ID NO:4) and oligodeoxynucleotide-EDTA 6-17 (SEQ ID NO:5) . The purine oligodeoxynucleotide with EDTA-Fe(II) at the 3' termini is located near the center of the major groove of the double-helical DNA antiparallel to the purine strand. The target site is located 0.42 kbp from the 3EP radiolabeled end of the restriction fragment.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a "triple helix" is defined as a double-helical nucleic acid with an oligonucleotide bound to a target sequence within the double-helical nucleic acid. The double-helical nucleic acid can be any double-stranded nucleic acid including double- stranded DNA, double-stranded RNA and mixed duplexes between DNA and RNA. Such double-helical nucleic acids preferably have a length greater than 500 bp, more preferably greater than 1 kb and most preferably greater than about 5 kb. In many applications, the double-helical nucleic acid comprises genomic DNA from procaryotic or eucaryotic sources. When such genomic DNA is used it can comprise fragmented DNA, DNA digested with restriction endonucleases, or DNA cleaved according to the methods of the invention or as described by Strobel and Dervan (1991) Nature 350:172-174. and Strobel, et al. (1991) Science 254:1639-1642. While such portions of genomic DNA are useful in practicing the invention, an important aspect of the invention is the formation of triple helices with chromosomal DNA either in situ or in vivo. When genomic DNA is utilized, the oligonucleotides used to form triple helices are particularly useful to detect the presence or absence of specific sequences within the genomic DNA for diagnostic and therapeutic purposes. Further, such genomic triple helices can be used in conjunction with the protocols of Strobel and Dervan, and Strobel, et al.. supra. for limited enzymatic cleavage of genomic DNA.

For example, numerous genetic diseases have been identified wherein a gene has been modified by way of substitution, insertion or deletion of one or more nucleotides to cause an inheritable recessive or dominant genotype. In addition, a number of polymorphisms, including restriction fragment length polymorphisms and other unique sequences, have been found to be associated with as yet unidentified inheritable gene defects such as these associated with autoimmune diseases and the like. Appropriate synthetic oligonucleotides can be used in the methods of the invention to detect such diagnostic sequences in genomic DNA (or in some cases in double-stranded cDNA derived from an appropriate tissue) as a means to diagnose the presence of one or more defective alleles for a particular disease, provided such diagnostic sequences are amenable to triple helix formation.

In addition, the triple helices of the invention have an enormous potential for the treatment of various disease states. For example, oligonucleotides can be selected which specifically bind to pathogenic double-stranded DNA including specific sequences required by pathogenic bacteria or viruses for replication or virulence. Alternatively, the oligonucleotide can be chosen to target a unique sequence of the pathogen which is not found in the genome of the pathogen's host. Such an oligonucleotide further includes a therapeutic agent which selectively kills the pathogen or a cell containing it based upon the selective specificity of the oligonucleotide for the pathogenic DNA.

Another important potential therapeutic application of triple helix technology involves cancer treatment by way of triple helix suppression of specific oncogenes including those of endogenous or viral origin. When so used, one or more oncogenes identified with a particular tumor type are used as targets for triple helix formation. Specific oligonucleotides designed for triple helix formation to suppress the expression of hyper-expressed oncogenes are designed to repress expression. Alternatively, when an activated oncogene contains unique sequences associated with such activation, oligonucleotides specific for the unique sequence and which contain a therapeutic agent can be used. Such therapeutic oligonucleotides are capable of selectively forming a triple helix with such sequences in those cancerous cells containing the activated oncogene thereby preferentially killing or repressing the cancer causing cell type.

As used herein, a "target sequence" within a double- helical nucleic acid comprises a sequence preferably greater than 10 nucleotides in length but preferably less than 20 nucleotides within the double-helical nucleic acid. The target sequence is most preferably between 11 to 18 bases. The target sequence, in general, is defined by the nucleotide sequence on one of the strands of the double-helical nucleic acid. In the preferred embodiments herein, the target sequence is defined by a purine-rich containing strand.

As used herein, a "purine-rich sequence" on one of the strands of double-helical nucleic acid is defined as a contiguous sequence wherein greater than 50% of the nucleotides of the target sequence contain a purine base. However, it is preferred that the purine-rich target sequence contain greater than 60% purine nucleotides, more preferably greater than 75% purine nucleotides, next most preferably greater than 90% purine nucleotides and most preferably 100% purine nucleotides. When such a target sequence contains greater than approximately 90% purine nucleotides, it is sometimes referred to as a purine tract or a substantially homopurine tract.

The oligonucleotides used in triple helix formation are generally of substantially the same length as the target sequence in the double-helical nucleic acid and have a sequence which permits binding of the oligonucleotide to the target sequence in either a parallel or antiparallel orientation as compared to the target sequence. In a parallel orientation, the oligonucleotide is oriented such that its 5' end is positioned at the 5' end of the target sequence and its 3' end is positioned at the 3• end of the target sequence. When oriented in an antiparallel orientation the 5' end of the oligonucleotide is positioned at the 3' end of the target sequence and the 3• end of the oligonucleotide is positioned at the 5' end of the target sequence. It is to be understood, of course, that reference to the 5' end and 3• end of the target sequence is not to be construed as the physical end of the double-helical nucleic acid but rather refers to the 5* to 3* sequence orientation in the strand containing the target sequence.

When the target sequence is a purine-rich sequence, parallel binding of the oligonucleotide occurs when the oligonucleotide is a pyrimidine-rich oligonucleotide. Antiparallel binding, however, occurs when a purine-rich oligonucleotide is used. As indicated hereinafter, the specific rules for parallel and antiparallel binding define a particular nucleotide contained within an oligonucleotide when the nucleotide at the "complementary position" in the target sequence is a specified nucleotide. This term means that when the oligonucleotide is positioned in a parallel or antiparallel orientation at a target sequence that there is a correspondence in the position of the various nucleotides in the oligonucleotide with the nucleotides contained in the target sequence. While it is believed that the oligonucleotide binding is within the major groove of a double-helical nucleic acid such as DNA and that the rules defining sequence binding have a physical basis with regard to the triplets proposed for triple helix formation, the use of such language is not to be construed as a limitation on the mechanism of triplex helix formation.

Parallel Binding to Purine-Rich Target Sequences

As indicated, parallel binding of an oligonucleotide to a purine-rich target sequence occurs when the oligonucleotide comprises a pyrimidine-rich oligonucleotide. In general, the following rules apply to the formation of triplets within the triple helix. The pyrimidine-rich oligonucleotide contains a T when the nucleotide at the complementary position in the purine-rich target sequence is A. Further, the pyrimidine-rich oligonucleotide contains a C when the nucleotide at the complementary position in the purine-rich target sequence is G. See Moser and Dervan (1987) Science 238:645-650.

Antiparallel Binding to Purine-Rich Target Seguences As indicated, antiparallel binding of an oligonucleotide to a purine-rich target sequence occurs when the oligonucleotide comprises a purine- rich oligonucleotide. In general, the following rules apply to formation of triplets in such a triple helix. The purine-rich oligonucleotide contains a G when the nucleotide at the complementary position in the purine-rich target sequence is G. Such an oligonucleotide also contains an A or T when the nucleotide in the complementary position in the purine-rich target sequence is A. See Beal and Dervan (1991) Science 251:1360-1363.

In addition to the foregoing rules, it has been determined that the nucleotide nebularine, or its analogs .e.g.. 2'-deoxynebularine) , is capable of binding to the pyrimidine nucleotide C in a purine- rich target sequence. This binding occurs when the nebularine is incorporated into an oligonucleotide designed for antiparallel binding to the purine-rich, but C-containing target sequence. When nebularine is used, it can be the sole nucleotide used to pair with a C nucleotide in the target sequence. However, since T is also capable of pairing with a C nucleotide in the purine-rich target sequence, albeit in a less energetically favorable manner, the oligonucleotide can contain nebularine nucleotides alone or nebularine nucleotides in combination with the pyrimidine nucleotide T.

When one or more nebularine nucleotides are used, the following rules apply. The purine-rich oligonucleotide contains a G when the nucleotide at the complementary position of purine-rich target sequence is G. In addition, the purine-rich oligonucleotide contains an A or a T when the nucleotide at the complementary position in the purine-rich target sequence is A. However, the total T content of the triple helix forming oligonucleotide is preferably less than 40% of the oligonucleotide sequence and most preferably less than 25%. When greater amounts of T nucleotides are used in an oligonucleotide, the antiparallel orientation of the oligonucleotide to the purine-rich target strand becomes less favorable and as a consequence, a shift to a parallel orientation can occur. Thus, an oligonucleotide containing 20 nucleotides designed for antiparallel orientation to a purine-rich target sequence of 20 nucleotides preferably contains no more than 5 to 8 T nucleotides to maintain the antiparallel orientation.

Production of Triple Helix Forming Oligonucleotides The oligonucleotides used in the invention to fdrm triple helices can be made synthetically by well- known synthetic techniques to contain a structure corresponding to the naturally occurring polyribonucleic or polydeoxyribonucleic acids.

Alternatively, the phosphoribose backbone of such oligonucleotides can be modified such that the thus formed oligonucleotide has greater chemical and/or biological stability. Biological stability of the oligonucleotide is desirable when the oligonucleotides are used in vivo for diagnostic or therapeutic uses. Such modified oligonucleotides are synthesized with a structure which is stable under physiological conditions which include enhanced resistance to nuclease degradation. Further, when used in vivo, such nucleotides preferably have a minimal length which permits targeted triple helix formation so as to facilitate the transport of the oligonucleotide across the membranes of the cytoplasm and nucleus.

In specific embodiments of this invention, a moiety is included in the triple helix forming oligonucleotide. Moieties such as a label, a therapeutic agent, or a cleavage moiety are incorporated along the length of any such oligonucleotide so as to provide precisely the detection, treatment or cleavage desired by the practitioner. Also, more than one moiety may be included in the oligonucleotide. Previously known and familiar synthesis protocols can be employed, in some cases using currently available automated technology, wherein such moieties can be incorporated into the triple helix forming oligonucleotide.

A nucleic acid-cleaving moiety can be attached to a nucleoside base during synthesis of a novel nucleoside and the so-modified nucleoside then incorporated into a selected oligonucleotide using standard procedures. This oligonucleotide containing the cleavage moiety recognizes the corresponding target sequence of a double-helical nucleic acid. For example, a metal chelator for cleaving a specific double-helical nucleic acid sequence is tethered to a triple helix forming oligonucleotide. Oligonucleotide-directed cleavage of double-helical DNA can be produced by a triple helix forming oligonucleotide DNA-EDTA-Fe probe. For example, thymidine can be replaced by thymidine with the iron chelator EDTA covalently attached at C-5. Reduction of dioxygen generates a localized hydroxyl radical at this point. Alternatively, the metal chelator may be attached to a selected nucleotide located within a given oligonucleotide sequence. In the presence of dioxygen (02) , an appropriate metal ion, and a reducing agent, the DNA-chelator probe yields a strand break at the target complementary DNA sequence, cleaving one or both strands at that site.

Oligonucleotides equipped with a DNA cleaving moiety have been described which produce sequence-specific cleavage of single-stranded DNA. See, e.g.. U.S. Patent No. 4,795,700. Examples of such moieties include oligonucleotide-EDTA-Fe probes (DNA-EDTA) which cleave a complementary single strand sequence (Dreyer and Dervan (1965) Proc. Natl. Acad. Sci. USA £32.:968; and Chu and Orgel (1965) Proc. Natl. Acad. Sci. USA £32.:963) . One example of a DNA-EDTA probe is a novel nucleoside, 5'-DMT-T*-triethylester derived from deoxyuridine to which is attached the metal chelator EDTA as described in detail in U.S. Patent No. 4,795,700. Such probes are also described in Dreyer and Dervan, Proc. Natl. Acad. Sci. USA, supra. These references disclose an EDTA-nucleoside composition incorporated into a 19-nucleotide base pair sequence of DNA complementary to a 19 bp sequence in a 167 bp restriction fragment of DNA from the plasmid pBR322. This DNA-EDTA probe was then used in the presence of the metal ion Fe(II) , atmospheric dioxygen, and the reducing agent dithiothreitol (DTT) to afford specific cleavage at its complementary 9 bp complement in single-stranded plasmid DNA.

Chelators or other cleavage moieties, as well as marker labels and therapeutic agents may also be incorporated into the triple helix forming oligonucleotide of the present invention at various positions for which the chemistry for attachment at such positions is known, provided that such attachment is accomplished so as not to disrupt the hydrogen-base pair bonding between the DNA or RNA sequences during triple helix formation. The triple helix forming oligonucleotide may be labeled in various well known ways for detection and diagnostic applications. For example, with radioactive metals such as wTc following the procedures of Elmalch, D.R., et al. (1984) Proc. Natl. Acad. Sci. USA £1:9 8 and EDTA or with fluorescent elements such as the lanthanides Tb+3 or Eu+3. Leung, et al. (1977) Biochem. Biophys. Res. Comm. 7.5:15. If a chelator is desired to be used in a cleavage moiety, other metal chelators may be used in place of EDTA such as polyamines or other chelators capable of binding Fe(II-III) or Cu(I-II) . Other polyamino carboxylic metal chelators may be utilized in place of EDTA such as 1,2-diamino- cyclohexane tetraacetic acid, diethylenetri-amine pentaacetic acid, ethylenediamine di-(-0- hydroxyphenol-acetic acid) , and hydroxyethylene diamine triacetic acid. A metal chelator may be attached to the nucleotide probe during synthesis via a hydrocarbon-amide linkage which may consist of several carbon atoms. The specificity of the probe for the reaction site is prescribed by the nucleotide sequence within which the metal chelator or other cleavage moiety is attached. The moiety can be incorporated into polydeoxyribonucleotides or polyribonucleotides of any desired length and sequenced using routine phosphoramidite or phosphotriester procedures.

One convenient synthesis of DNA-EDTA probes involves the incorporation of a modified thymidine into an oligonucleotide by chemical methods. This approach allows for automated synthesis and affords control over the precise location of the EDTA moiety at any thymidine position in the oligonucleotide strand, Felsenfeld, et al. (1967) Annu. Rev. Biochem. 36:407. In an embodiment of this invention, bifunctional DNA- EDTA probes are used for recognition and cleavage of a double-stranded nucleic acid. These probes allow triple helix formation at a discrete location to be mapped on large DNA using gel electrophoresis. The preferred general conditions for the cleavage reactions are as follows: approximately 100 nM in bp radio labeled restriction fragment (approximately 10,000 cpm) , 25 nM tris/acetate, pH 7.0, 1 nM sper ine, (MY) , 100 nM NaCl, 100 umolar in vp sonicated, deproteinized cath-thimus DNA, 20 volume- percent ethylene glycol, 1 μmolar DNA EDTA probe, 25 μmolar Fe(II) and 2 nM DTT. The cleavage reactions were run for approximately 16 hours at 0-25°C. These conditions may be varied without departing from the scope of this invention.

As described in the examples below, the affinity cleaving method utilizing DNA, EDTA and known in the art allows the effect of reaction conditions, probe length, and single base mismatches on triple helix formation to be analyzed on high resolution sequencing gels. Precise methods for quantitation and measurement and determination of the presence and orientation of triple helices is set out in more detail in the Examples below.

As will be seen in the Examples, the directional orientation of the third strand as well as the identity of the grooves in right-handed DNA-helix occupied by the bound DNA-EDTA probe can be analyzed by high resolution gel electrophoresis.

Additionally, the location of triple helices within large pieces of DNA can be mapped by double-strand breaks analyzed by nondenaturing agarose gel electrophoresis. Synthesis and the preparation of necessary and desired component parts of the probes of the present invention, and their assembly is believed to be within the duties and tasks performed by those with ordinary skill in the art and, as such, are capable of being performed without undue experimentation.

The oligonucleotide probes of the present invention are not limited to the production of sequence- specific cleavage of double-stranded DNA by triple helix formation, but may also be utilized as diagnostic agents when a radioisotope labeled, fluorescing, or otherwise detectable metal ion is attached to the probe. The probes of the present invention may also be used as target-specific therapeutics with the attachment of an "artificial" or natural gene repressor or other effective agent to the oligonucleotide.

The following is presented by way of example, and not to be construed as a limitation to the scope of the appended claims.

EXAMPLE 1

This example describes the design of heterocycles for the recognition of CG Watson-Crick base pairs within the pur*pur«pyr triple helical motif. Model building studies suggested that the deoxyribonucleoside 2'- deoxynebularine (N) would fulfill this role (Figure 1). It was assumed that the purine core of 2'- deoxynebularine would mediate base stacking interactions in the third strand. In addition, the heterocyclic ring system provides a hydrogen bond acceptor (Nl) which should allow the formation of one hydrogen bond to the exocyclic amino group of cytosine or adenine (Figure 1) .

The affinity cleaving method was used to analyze the binding of 2*-deoxynebularine (N) to all four possible combinations of the two Watson-Crick base pairs. It was found that within the particular DNA sequence studied N interacts preferential with CG base pairs. The new recognition element was employed to target a single site of the HIV genome containing two CG base pairs within plasmid DNA.

Deoxynebularine phosphoramidite was purchased from Glen Research. All other phosphoramidites and chemicals for DNA synthesis were obtained from Applied Biosystems Inc. Restriction endonucleases and all other enzymes were purchased from Boehringer- Mannheim, New England Biolabs or Sigma. The Sequenase™ DNA sequencing kit (Version 2.0) was obtained from United States Biochemical Inc. Deoxynucleoside triphosphates (100 mM solutions) , calf thymus DNA and Nick™-columns were purchased from Pharmacia LKB. The radiolabeled triphosphates 5»-(α-32P)dGTP (> 3000 Ci/mmol) , 5'(γ32p) ATP(> 5000 Ci/mmol) and 5'-(α-35S)dATP (> 1000 Ci/mmol) were obtained from Amersham. All other chemicals were of analytical or HPLC grade.

Whenever possible, standard molecular biological methods were used (Sambrook, J. , Fritsch, E. F. , Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.). Agarose gel electrophoresis was performed in 1 x TAE buffer and polyacrylamide gel electrophoresis was carried out in 1 x TBE buffer (Sambrook et al.. ibid.) . Cerenkov radioactivity was measured with a Beckman LS 2801 scintillation counter. Oligodeoxynucleotides were synthesized on an Applied Biosystems Model 3808 DNA synthesizer using β- cyanoethyl phosphoramidite triester chemistry (Beaucage, S. L. , and Caruthers, M. 1-1. (1981) Tetrahedron Lett. 22:1859-1862; Sinha, N. D. ,

Biernat, J., McManus, J., Koster, H. (1984) Nucleic Acids Res. 12.:4539-4557) The nucleoside analog T* was prepared according to published procedures and was incorporated at the 3' end of oligodeoxynucleotides via the 5'-O-DMT-thymidine-

EDTA-triethylester-3'-succinyl controlled pore glass (Dreyer, G. B. , and Dervan, P. B. (1985) Proc. Natl. Acad Sci. USA 82:968-972.. Unmodified oligodeoxynucleotides were deprotected under standard conditions using concentrated ammonium hydroxide. Oligodeoxynucleotides containing the nucleoside analog T* were treated with 0.1 N NaOH (1.5 mL) at 55βC for 24 hours. The DMT protected oligo ers were purified by reverse phase FPLC chromatography (Pharmacia ProRPC 15 mm HR 10/10; gradient of 0-40% acetonitrile in 100 mM triethylammonium acetate (pH=7.0)). Lyophilized fractions were treated (20 min, 23βC) with an 80% solution of acetic acid in water (500 μl) in order to remove the DMT protecting group. The oligomers were then repurified by FPLC. The concentrations of the oligodeoxynucleotides were determined by UV measurements (A260) , using the following molar extinction coefficients: 15400 (A) , 11700 (G) , 7300 (C) , 8800 (T and T*) , and 6000 (N) cm"1M*1. After lyophilization the oligodeoxynucleotides were stored dry at -78βC.

Analytical HPLC was performed with a Hewlett-Packard 1090 Liquid Chromatograph using a reverse phase VYDAC 201HS54 4.6 mm x 25 cm 5 micron C18 column. The purified oligodeoxynucleotides (3 nmol) were digested simultaneously with snake venom phosphodiesterase (3 μL, 2.4 μg/μL) and calf intestine alkaline phosphatase (3 μL, 1 U/μL) in 50 mM Tris-HCl (pH 8.1), 100 mM MgCl2. The reaction mixture was incubated at 37 °C for 3h, filtered through a 0.45 μm Nylon-66 syringe filter (Rainin) and lyophilized. The sample was dissolved with 10 μL of 10 mM ammonium phosphate (pH=5.1 )/8% methanol buffer, and an aliquot of the solution was injected onto the C18 reverse phase column. The products were eluted with 10 mM ammonium phosphate (pH=5.1)/8% methanol and detected at A260. Comparison with standard solutions • of A, T and N established the composition of the oligodeoxynucleotides.

A finity Cleaving Reactions of 39mer Duplex Targets For the preparation of the duplex targets, each single-stranded oligodeoxynucleotide (100 pM) of sequence composition 5•d(AgTj(CT)3A5G3XG4AG4AG3A5(CT)3)3• (X=A, G, C, or T) (SEQ ID NO:2) was 5' end labeled using T4 polynucleotide kinase and γ-32P ATP according to standard procedures (Sambrook et al.. supra.) . The reaction mixture was twice extracted with TE buffer-saturated phenol (1.0 volume) and twice extracted with 24:1 chloroform/isoamyl alcohol (1.0 volume). The DNA was ethanol precipitated, and the radiolabeled oligodeoxynucleotides were annealed to their unlabeled complementary oligodeoxynucleotides. The resulting duplexes were purified on 15% nondenaturing polyacrylamide gels (19:1; monomer/bis). Gel bands were visualized by autoradiography and desired bands were excised from the gel, crushed and eluted with 1 mL 200 mM NaCl at 37 °C for 20 h. The eluents were passed through 0.45 μm Centrex filters and lyophilized. The residue was taken up in 100 μL distilled water and the solution was then desalted by passing it through a Nick™- column. The radiolabeled duplex-DNA was finally isolated by ethanol precipitation. Specific DNA cleavage reactions for adenine were performed as described previously (Iverson, B. L. , and Dervan, P. B. (1987) Nucleic Acids Res. 15:7823- 7830) . The affinity cleaving reactions were executed in a total volume of 80 μL by combining a mixture of oligodeoxynucleotide-EDTA (100 nM) and Fe(NH4)2(S04)2 • 6H20 (250 nM) with the 3P-labeled duplex ("120 000 cpm) in a solution of tris-acetate (50 mM, pH=7.4 at 23 °C), NaCl (20 mM) , spermine (100 μM) and calf thymus DNA (100 μM in base pairs). The oligodeoxynucleotide probe was allowed to equilibrate with the DNA duplex target at 37 βC for 4 hr. The cleavage reactions were then initiated by the addition of dithiothreitol (4 mM final concentration) and allowed to proceed at 37 "C for 14 h. The reactions were quenched by freezing (liquid N2) followed by lyophilization. The residue was suspended in 10 μL formamide loading buffer (90% formamide, 10% 10 x TBE buffer, 0.02% bromophenol blue, 0.02% xylene cyanol) and transferred to new tubes. The DNA suspensions were assayed for specific activity by scintillation counting and diluted to 5000 cpm/μL. The cleavage products were denatured at 90βC for 5 min and 4 μL of each sample were separated by 20% denaturing polyacrylamide gel electrophoresis (19:1; monomer/bis). The gels were exposed to X-ray film (Amersham Hyperfilm™-MP) at -78 βC with a single intensification screen or to a storage phosphor screen.

Quantitation of Cleavage Efficiencies by Storage Phosphor Technology Autoradiography

The relative cleavage efficiencies were determined by quantitation on a Molecular Dynamics 400S

Phosphorimager. Gels were exposed to the storage phosphor screen (Kodak storage screen S0230 obtained from Molecular Dynamics) in the dark at 23 °C for 4 h

(Johnston, R. F. , Pickett, S. C, and Barker, D. L.

(1990) Electrophoresis .11:355-360). τhe data were analyzed using the I ageQuant v. 3.0 software. The radiation background of the screen was determined by performing volume integrations over four independent reference sites. All other volume integrations were based on the averaged background value obtained.

Integration of the cleavage bands was performed over the five most efficiently cleaved nucleotides. Rectangles of the same size were used for each lane and the amount of radioactivity found in the respective untreated control lane was subtracted from the obtained values. The relative cleavage efficiencies were evaluated by calculating the ratio of the radioactivity of the site-specific cleavage band by the integrated volume of the entire lane. The given values represent the average over two independent experiments.

Construction of the Plasmid pULHIV

The plasmid pULHIV was obtained by cloning the oligodeoxynucleotides 5*d(A2T2CG2C- A2GAG2CGAG4CG2CGACT)3' (SEQ ID NO:6) and

5'd(CTAGAGTCGC2GC4TCGC2TCT2GC2G)3l (SEQ ID NO:7) into the large EcoRI/Xbal restriction fragment of pUC19 by using T4 DNA Ligase. The ligation mixture was employed to transform Epicurian™ Coli XL1 Blue competent cells (Stratagene) . The cells were grown on Luria Bertani medium agar plates containing 100 μg/mL ampicillin, X-gal, and IPTG. Large scale plasmid isolation of appropriate clones was performed using QIAGEN purification kits (Diagen) according to the manufacturer's protocol. The sequence of the insert was subsequently confirmed by dideoxynucleotide sequencing (Sanger, F. , Nicklen, S., and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 72.:2251-2255) using the #1201 M13 reverse sequencing primer (New England Biolabs) . Affinity Cleaving Reactions of the pULHIV EcoO1091-SspI Restriction Fragment

The pULHIV Eco01091-Sspl restriction fragment was produced as follows. Plasmid DNA (20 μg) was linearized with Eco01091 and then end labeled with

5'-(α-32P)dGTP employing sequenase™ (version 2.0) as the enzyme. The reaction mixture was applied to a

Nick™-column to remove unincorporated radiolabeled nucleotide triphosphates. The labeled DNA was then ethanol precipitated and digested with Sspl. The resulting 3'-32P-end labeled fragment (2515 bp) was purified by agarose gel electrophoresis (1% Nusieve GTG Agarose, FMC) . Gel bands were visualized by autoradiography, the desired band was excised from the gel and transferred to an eppendorf tube. The agarose was frozen and thawed (3x) and the resulting suspension was centrifuged (20 min, 14K) . The supernatant was removed and was twice extracted with TE buffer saturated phenol (1.0 voiume) and twice extracted with 24:1 chloroform/isoamyl alcohol (1.0 volume) . The DNA was ethanol precipitated, dissolved in 100 μL TE buffer and then passed through a Nick™- column. Finally the DNA was ethanol precipitated and dissolved in water to a final concentration of 10000 cpm/μL.

The affinity cleaving reactions were executed in a total volume of 80 μL by combining a mixture of oligodeoxynucleotide-EDTA (2 μM) and Fe(NH4)2(S04)2»6H20(5 μM) with the 32P-labeled restriction fragment ("40 000 cpm) in a solution of tris-acetate (50 mM, pH 7.4 at 23βC), NaCl (10 mM) , spermine (1 mM) and calf thymus DNA (100 μM in base pairs) . The oligodeoxynucleotide probe was allowed to equilibrate with the DNA duplex target at 37 °C for 4 h. The cleavage reactions were then initiated by the addition of dithiothreitol (4 mM) and allowed to proceed at 37 βC for 12 h. The reactions were stopped by precipitation of the DNA with ethanol. The residue was resuspended in TE buffer (30 μL) and transferred to new tubes. The DNA suspensions were assayed for specific activity by scintillation counting and diluted to 5000 cpm/20 μL. An aliquot (10 μL) of glycerol gel loading buffer (30% glycerol in water, 0.25% bromophenol blue, 0.25% xylene cyanol) was added to 20 μL of each of the samples. The cleavage products were separated by 5% nondenaturing polyacrylamide gel electrophoresis (19:1 monomer/bis). The gel was dried on a slab dryer and visualized by autoradiography (Amersham HyperfilmTH-MP, -78°C, intensification screen) .

Synthesis of Oligodeoxynucleotides Containing 2'-Deoxynebularine and Base Composition Analysis

Oligodeoxynucleotides 1-17 (SEQ ID NOS:l and 5) were synthesized by solid phase methods using /.-cyanoethyl phosphoramidite chemistry (Beaucage, S. L. , amd

Caruthers, M. 1-1. (1981) Tetrahedron Lett. 22:1859- 1862; Sinha, N. D. , Biernat, J. , McManus, J. , Koster, H. (1984) Nucleic Acids Res. 12:4539-4557). The 21- deoxynebularine phosphoramidite coupled as efficiently as the A, G, c, and T phosphoramidites. The base composition of oligodeoxynucleotides 1, 6 (SEQ ID NO:l), 11, 16, and 17 (SEQ ID N0:2) containing 2•-deoxynebularine were established by HPLC analysis. For this, the oligodeoxynucleotides were treated with snake venom phosphodiesterase and calf intestine phosphatase. The nucleoside monomers obtained were separated by HPLC and identified by their HPLC retention times and UV spectra. Comparison of the integrated areas of the HPLC peaks with that of standard solutions of A, T and N confirmed the correct base composition of the oligodeoxynucleotides.

Analysis of Binding Specificity The relative affinity of 2 •-deoxynebularine for all four Watson-Crick base pairs within a pur«pur»pyr triple-helix motif was examined by affinity cleaving (Dreyer and Dervan, 1985, supra. ) . A series of 15 nt oligodeoxynucleotides 1-5 (SEQ ID N0:1) (Figure 2), differing at one base position 5'd(TG4TG4ZG3T*) 3 ' (SEQ ID N0:1) (Z = N, A, G, C, or T) ; and equipped with the DNA cleaving moiety, thymidine-EDTA»Fe(II) (T*) at a single thymidine position at the 3 * end was prepared. The relative stabilities of the triple helical structures formed upon complexation of these oligodeoxynucleotides with 39-bp DNA duplexes containing one variable base pair site

Figure imgf000025_0001
5'd(CT(AG)4T5C3TC4TC4YC3T5(AG)3)3' (SEQ ID NO:2 • SEQ ID NO:3) (XY = AT, CG, GC, or TA) were then measured. The DNA affinity cleaving reactions were performed under conditions which allowed the difference in stability between single base triplets to be distinguished (100 nM oligodeoxynucleotide-EDTA, 100 μM spermine, 20 mM NaCl) . The most efficient cleavage was observed for the combinations Z = A or T, XY = AT, and Z = G, XY = GC (results not shown) . These cleavage patterns reflect the known ability of G, A, and T to form G»GC, A»AT, and T»AT base triplets, respectively (Beal, P. A., and Dervan, P. B. (1991) Science 251:1360-1363). Importantly, intense cleavage was also detected for a N*CG triplet (results not shown) . The base triplets NΑT, C»AT, T»CG, and A»GC produced moderate cleavage. Only weak cleavage was observed for the 12 additional triplet combinations. The relative cleavage intensities were determined by quantitative storage phosphor autoradiography and are presented as histograms in Figure 3.

Site-specific double-stranded cleavage of plasmid DNA. The plasmid pULHIV was prepared in order to determine whether 2'-deoxynebularine can be used to recognize CG base pairs within a larger fragment of double-helical DNA. For this, the purine-rich target sequence 5'd(AGAG2CGAG4CG2)3• • δ'dfC^G^TCGC^TCTJS ' (SEQ ID NO:8), a sequence which occurs naturally in the HIV genome (Ratner, L. , et al. (1985) Nature 313:277-284) , was cloned into pUC19 DNA. The ability of oligodeoxynucleotides 6-17 (SEQ ID NO:5) (Figure 4) to bind specifically to the target sequence was examined by affinity cleaving. Conditions sensitive to the stability of the base triplet at the CG sites (2 μM oligodeoxynucleotide-EDTA, 1 mM spermine, 10 mM NaCl) were used. A 2.51 kbp EcoO1091-SspI restriction fragment of pULHIV, which contains the target sequence, located 0.42 kbp from the 32P radiolabeled end (Figure 13) , was isolated. This restriction fragment was allowed to react with 5'd(G2Z2G4ZlGZ2G2ZlGT*)3* oligodeoxynucleotides- EDTA-Fe(II) 6-17 (SEQ ID NO:5) , which differ at four variable positions (Zl = A, T or N; Z2 = N, A, G, C, or T) , in the presence of dithiothreitol at 37 °C (pH 7.4). The cleavage products were separated by 5% nondenaturing polyacrylamide gel electrophoresis. One major cleavage product, 0.42kbp in size, indicated sequence specific cleavage was only observed for the oligodeoxy-nucleotides 6 (Zl = A; Z2 = N) , 10 (Zl = A; Z2 = T) , 11 (Zl = T; Z2 = N) , and 15 (Zl = T; Z2 = T) , respectively (results not shown) .

It is to be understood that various other modifications will be apparent to and can readily be made by those skilled in the art, given the disclosure herein, without departing from the scope and spirit of this invention. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the description as set forth herein, but rather that the claims be construed as encompassing all the features of patentable novelty which reside in the present invention, including all features which would be treated as equivalents thereof by those skilled in the art to which this invention pertains.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: California Institute of Technology, Pasadena, California 91125, U.S.A. (ii) TITLE OF INVENTION: Triple helix recognition of DNA

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Richard F. Trecartin (B) STREET: 4 Embarcadero Center, Suite 3400

(C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94111 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Herewith

(C) CLASSIFICATION: Not yet assigned

(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 07/850,503

(B) FILING DATE: 13-MAR-1992

(C) CLASSIFICATION: 435

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Trecartin, Richard F (B) REGISTRATION NUMBER: 31,801

(C) REFERENCE/DOCKET NUMBER: FP-56557/RFT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (415) 781-1989

(B) TELEFAX: (415) 398-3249

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (ix) FEATURE :

(A) NAME/KEY: modified_base

(B) LOCATION: 15

(D) OTHER INFORMATION: /note= "EDTA is covalently attached at C-5."

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 11

(D) OTHER INFORMATION: /note- "Base is A or C or G or T or 2'-deoxynebularine."

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

TGGGGTGGGG NGGGT 15

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME KEY: misc_binding

(B) LOCATION: 5..43

(D) OTHER INFORMATION: /note- "Sticky single-stranded end from base 1 to 4. Double stranded from base 5 to 43 to complementary strand, SEQ ID NO:3."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

AATTCTCTCT AAAAAGGGNG GGGAGGGGAG GGAAAAACTC TCT 43

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 43 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: both

(D) TOPOLOGY: linear

(ii) MOLECULE. TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_binding (B) LOCATION: 5..43 (D) OTHER INFORMATION: /note= "Sticky single-stranded end from base 1 to 4. Double-stranded from base 5 to 37 to complementary strand, SEQ ID N0:2."

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CTAGAGAGAG TTTTTCCCTC CCCTCCCCNC CCTTTTTAGA GAG 43

(2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: GTCGCCGCCC CTCGCCTCTT GCCGAAT 27

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: modified_base (B) LOCATION: 15

(D) OTHER INFORMATION: /note- "EDTA covalently attached at C-5."

(ix) FEATURE:

(A) NAME KEY: modified_base (B) LOCATION: 13

(D) OTHER INFORMATION: /note- "Base is A or T or 2' -deoxynebularine."

(ix) FEATURE:

(A) NAME KEY: modified_base (B) LOCATION: 10

(D) OTHER INFORMATION: /note- "Base is A or G or C or T or 2'-deoxynebularine."

(ix) FEATURE:

(A) NAME/KEY: modifiedjase (B) LOCATION: 8

(D) OTHER INFORMATION: /note= "base is A or T or 2'-deoxynebularine. "

(ix) FEATURE: (A) NAME/KEY: modified_base

(B) LOCATION: 3

(D) OTHER INFORMATION: /note- "Base is A or G or C or T or 2'-deoxynebularine. "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGNGGGGNGN GGNGT 15

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AATTCGGCAA GAGGCGAGGG GCGGCGACT 29 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTAGAGTCGC CGCCCCTCGC CTCTTGCCG 29

(2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AGAGGCGAGG GGCGG 15

Claims

WHAT IS CLAIMED IS:
1. A triple helix comprising a large double-helical nucleic acid and an oligonucleotide bound to a purine-rich target sequence within said double- helical nucleic acid wherein said purine-rich target sequence contains one or more or the pyrimidine nucleotide C and said oligonucleotide contains a nebularine nucleotide when the nucleotide at the complementary position in said purine-rich target sequence is C.
2. The triple helix of Claim 1 wherein said oligonucleotide is bound to said purine-rich target sequence in an antiparallel orientation.
3. The triple helix of Claim 2 wherein said oligonucleotide is a purine-rich oligonucleotide.
4. The triple helix of Claim 3 wherein said oligonucleotide contains G when the nucleotide at the complementary position in said purine-rich target sequence is G and an A or a T when the nucleotide at the complementary position in said purine-rich target sequence is an A.
5. The triple helix of Claim 1 wherein said oligonucleotide bound to said target sequence within said double-helical nucleic acid further comprises at least one nucleotide to which a moiety is attached.
6. The triple helix of Claim 5 wherein said moiety is a detectable moiety.
7. The triple helix of Claim 5 wherein said moiety is a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.
8. The triple helix of Claim 5 wherein said moiety comprises a therapeutic agent.
9. A synthetic triple helix forming oligonucleotide capable of binding in an antiparallel orientation to a purine-rich target sequence in a large double- helical nucleic acid comprising an oligonucleotide containing a nebularine nucleotide when the nucleotide at the complementary position in said purine-rich target sequence is C.
10. The synthetic triple helix forming oligonucleotide of Claim 9 wherein said oligonucleotide further comprises a G when the nucleotide in the complementary position in said purine-rich target sequence is G and an A or T when the nucleotide at the complementary position of said purine-rich target sequence is A.
11. The synthetic oligonucleotide of Claim 9 wherein said oligonucleotide further comprises at least one nucleotide to which is attached at least one moiety.
12. The synthetic oligonucleotide of Claim 9 wherein said moiety is a detectable moiety.
13. The synthetic oligonucleotide of Claim 9 wherein said moiety is a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.
14. The synthetic oligonucleotide of Claim 9 said moiety comprises a therapeutic agent.
15. A process for forming a triple helix wherein an oligonucleotide is bound to a purine-rich target sequence within a large double-helical nucleic acid, said target sequence containing one or more of the pyrimidine nucleotide C, said method comprising contacting a large double-helical nucleic acid with an oligonucleotide capable of binding to said target sequence contained within said double-helical nucleic acid, said oligonucleotide containing a nebularine nucleotide when the nucleotide at the complementary position in said purine-rich target sequence is c.
16. The process of Claim 15 wherein said oligonucleotide is bound to said purine-rich target sequence in an antiparallel orientation.
17. The process of Claim 16 wherein said oligonucleotide is a purine-rich oligonucleotide.
18. The process of Claim 17 wherein said oligonucleotide contains G when the nucleotide at the complementary position in said purine-rich target sequence is G and an A or a T when the nucleotide at the complementary position in said purine-rich target sequence is an A.
19. The process of Claim 15 wherein said oligonucleotide bound to said target sequence within said double-helical nucleic acid further comprises at least one nucleotide to which a moiety is attached.
20. The process of Claim 19 wherein said moiety is a detectable moiety.
21. The process of Claim 19 wherein said moiety is a cleaving moiety capable of causing cleavage of said double-helical nucleic acid.
22. The process of Claim 19 wherein said moiety comprises a therapeutic agent.
PCT/US1993/002352 1992-03-13 1993-03-11 Triple helix recognition of dna WO1993018187A1 (en)

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US6451968B1 (en) 1991-05-24 2002-09-17 Isis Pharmaceuticals, Inc. Peptide nucleic acids
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6451968B1 (en) 1991-05-24 2002-09-17 Isis Pharmaceuticals, Inc. Peptide nucleic acids
US6441130B1 (en) 1991-05-24 2002-08-27 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
EP0672193A4 (en) * 1992-09-29 1995-11-22 Isis Pharmaceuticals Inc Oligonucleotides having a conserved g 4? core sequence.
US5952490A (en) * 1992-09-29 1999-09-14 Isis Pharmaceuticals, Inc. Oligonucleotides having a conserved G4 core sequence
EP0672193A1 (en) * 1992-09-29 1995-09-20 Isis Pharmaceuticals, Inc. Oligonucleotides having a conserved g 4? core sequence
EP0864002A1 (en) * 1995-10-27 1998-09-16 Elliot R. Ramberg Methods and compositions for detection of specific nucleotide sequences
EP0864002A4 (en) * 1995-10-27 1999-07-28 Elliot R Ramberg Methods and compositions for detection of specific nucleotide sequences
DE19806962B4 (en) * 1997-02-22 2004-08-05 Universität Heidelberg Labeling of nucleic acids with specific sample mixtures
JP2005527181A (en) * 2001-07-20 2005-09-15 インジェネウス コーポレイションIngeneus Corp. Parallel or antiparallel homologous or complementary binding of nucleic acids or their analogues to form duplex, triplex or quadruplex complexes.
US7199228B2 (en) * 2001-08-17 2007-04-03 Bioniche Life Sciences, Inc Oligonucleotide compositions and their use to induce apoptosis
US7893242B2 (en) * 2001-08-17 2011-02-22 Bioniche Life Sciences Inc. Oligonucleotide compositions and their use to induce apoptosis
US8350016B2 (en) 2001-08-17 2013-01-08 Bioniche Life Sciences Inc. Oligonucleotide compositions and their use to induce apoptosis

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