WO1993012230A1 - Triple-helix formation at (punpyn).(punpyn) tracts - Google Patents

Triple-helix formation at (punpyn).(punpyn) tracts Download PDF

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Publication number
WO1993012230A1
WO1993012230A1 PCT/US1992/010792 US9210792W WO9312230A1 WO 1993012230 A1 WO1993012230 A1 WO 1993012230A1 US 9210792 W US9210792 W US 9210792W WO 9312230 A1 WO9312230 A1 WO 9312230A1
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nucleic acid
oligonucleotide
tract
purine
sequence
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PCT/US1992/010792
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French (fr)
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Sumedha D. Jayasena
Brian H. Johnston
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Sri International
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Priority claimed from US07/808,452 external-priority patent/US6063612A/en
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Publication of WO1993012230A1 publication Critical patent/WO1993012230A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6839—Triple helix formation or other higher order conformations in hybridisation assays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3511—Conjugate intercalating or cleaving agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the use of triple helical DNA in the control of gene expres-
  • the present invention describes third strand molecules that utilize both Pu*PuPy and Py «PuPy base triplets to form a continuous DNA triple-helix at tandem oligo-
  • the oligopyrimidine strand lies in parallel orientation to the oligopurine tract of the duplex (Moser & Dervan 1987; Praseuth et al., 1988) , and the pairing of T with AT and C + with GC makes the recognition sequence-specific. Because the C + "GC triplet re ⁇ quires protonated cytosines on the Hoogsteen (third) strand, triplex formation is facilitated by low pH (Mirkin et al., 1987).
  • Pu-PuPy In addition to PyPuPy triplets, another general class of base triplets, Pu-PuPy (GGC and A"AT) , has also been proposed to explain the sequence-specific recognition of oligopurineoligopyrimidine tracts in duplex DNA by oligopurine single strands ( Figure la(ii)). In this case the oligopurine single strand, which again lies in the major groove, is antiparallel to the oligopurine tract in duplex DNA
  • DNA recognition via triplex forma ⁇ tion has been restricted to oligopurineoligopyri i- dine tracts using single strands consisting exclu- sively of either purines or pyrimidines.
  • the following approaches (Home & Dervan, 1990) have been proposed for recognition of all four bases in triplex formation: (1) synthesizing novel, unnatu ⁇ ral bases to complete the triplet code; (2) exclud- ing the recognition of certain base pairs in a triplex motif by incorporating abasic sites in the single strand, which may decrease the specificity of triplex formation; and (3) designing homopyrimidine single strands capable of binding to alternate strands of duplex DNA.
  • polarity considerations required the design of an oligonucleotide with an unnatural 3 '-3 ' linkage having the proper spacing between two oligopyrimi ⁇ dine strings (Home & Dervan, 1990) .
  • oligonucleotide capable of forming a triple- helix with a nucleic acid molecule containing a target nucleic acid sequence is described, where the oligonucleotide is comprised of tandem, alternating tracts of purine and pyrimidine sequences, and where the oligonucleotide is effective to form a triple- helix structure with a target nucleic acid sequence having alternating tracts of purine and pyrimidine sequences.
  • the oligonucleotide of the present invention typically contain at least one purine tract adjacent at least one pyrimidine tract.
  • the target sequence, which oligonucleotide binding is directed toward usually contains at least about 8 nucleo- tides.
  • the nucleic acid target sequences of the present invention can be duplex DNA molecules, single-strand DNA molecules, and/or RNA molecules. Typically, when the nucleic acid molecule is a single stranded nucleic acid molecule, and the oli- gonucleotide contributes two of the three strands of the triple-helix.
  • the oligonucleotide further includes at least one moiety attached to the oligonucleotide, where the moiety is capable of cleaving the DNA molecule containing the target nucleic acid sequence.
  • a representative group of such cleaving moieties includes the follow- ing: phenanthroline Cu(II) , Zn(II), Fe(II)-EDTA, Cu(II)-bipyridine, and Cu(II)-terpyridine.
  • Also described is a method of cleaving a nucleic acid molecule containing a target nucleic acid sequence wherein the nucleic acid molecule is contacted with an oligonucleotide comprised of tandem, alternating tracts of purine and pyrimidine sequences, where the oligonucleotide is effective to form a triple-helix structure with a target nucleic acid sequence having alternating tracts of purine and pyrimidine sequences, and where the oligonucleo ⁇ tide includes at least one moiety which is capable of cleaving the nucleic acid molecule containing the target nucleic acid sequence.
  • the oligonucleotide concentration is effective to produce cleavage of the nucleic acid molecule containing the target nucleic acid sequence
  • the target nucleic acid can be single- or double- stranded; this includes DNA and RNA targets.
  • Figure 1 A. Schematic illustration of different triplex motifs, (i) A PyPuPy triple helix in which the third, oligopyrimidine strand is parallel to the purine strand of the duplex. (ii) A PuPuPy triple helix in which the third, oligopu ⁇ rine strand is antiparallel to the purine strand of the duplex.
  • the third-strand region shown in the middle in gray and black-and-white stripes
  • the purine tract of the third strand forms base pairs (black bars) with the purine tract of one strand of the Watson-Crick hairpin (making Pu-PuPy base triplets)
  • the pyrimidine tract of the third strand black-and— white
  • White bars represent Watson-Crick hydrogen bonding
  • arrows indicate the polarity (5' to 3') of the strand.
  • Figure 2. This figure illustrates the KMn0 4 reactivity of oligonucleotide I (SEQ ID N0:1) ( Figure 8) .
  • FIG. 3 This figure illustrates the KMn0 4 ⁇ ni DEpc reactivity of oligonucleotide II (SEQ ID NO:2) ( Figure 8) .
  • FIG. 4 This figure illustrates the KMn0 4 Md DEPC reactivity of oligonucleotide III (SEQ ID NO:3) ( Figure 8) .
  • FIG. 5 This figure illustrates the KMn0 4 tnd DEPC reactivity of oligonucleotide IV (SEQ ID NO:4) ( Figure 8) .
  • FIG. 6 This figure illustrates the KMn0 4 DEP c. and DMS reactivity of oligonucleotide V (SEQ ID NO:5) ( Figure 8) .
  • Figure 7. This figure summarizes the reactivi ⁇ ty patterns and deduced secondary structures for each of oligonucleotides I-V: Closed arrows, KMn0 4 reactivity that is not sensitive to Mg ++ ions; the size of the arrow roughly correlates with the extent of base reactivity. (The arrow shown in parentheses is a presumably reactive thymine that was run off the gel.) Open arrows, KMn0 4 reactivity of thymines sensitive to the presence of Mg ++ ions.
  • Figure 8 This figure presents the nucleotide sequences of oligonucleotides I-V.
  • Figure 9 illustrates three potential sites for targeting within the LTR region of HIV-1 with single-stranded oligonucleotides.
  • Figure 10 presents the sequences of oligonucle- otides Al (SEQ ID NO:9) and A2 (SEQ ID N0:10), Bl (SEQ ID NO-.ll) and B2 (SEQ ID NO:12), Cl (SEQ ID NO:13) and C2 (SEQ ID NO:14) which are designed to target sequences of Figure 9A, 9B, and 9C, respec ⁇ tively.
  • Next to each of these oligonucleotides is the general pattern of base triplets expected to form when triplexes are formed.
  • Oligonucleotides K (SEQ ID NO:15), L (SEQ ID NO:16), and M (SEQ ID NO:17) are oligonucleotides used in control experi ⁇ ments.
  • Figure 11 illustrates the chemistry of the attachment of a phenanthroline moiety to a cysteine- containing polypeptide.
  • Figure 12A illustrates three TDA target sites (D, SEQ ID NO:18; E, SEQ ID NO:19; F, SEQ ID NO:20) within the 5786-8887 nucleotide region of the HIV-1 genome.
  • Figure 12B shows a schematic representation of triplex formation at target mRNA sequences D, E, and F. For each mRNA sequence two oligonucleotides
  • oligonucleotides designed to function as conventional antisense agents.
  • X indicate abasic sites of oligonucleotides
  • Figure 13 provides an overview of a method for targeting and inactivation of HIV mRNA using TDA oligonucleotides.
  • Figure 14A shows oligonucleotide sequences containing the target sequences for Example 4B. These sequences are also presented as: Target sequence #1, SEQ ID NO:30 and SEQ ID NO: 31; Target sequence #2, SEQ ID NO:32 and SEQ ID NO:33; Target sequence #3, SEQ ID NO:34 and SEQ ID NO:35; Target sequence #4, SEQ ID NO:36 and SEQ ID NO:37; Target sequence #5, SEQ ID NO;38 and SEQ ID NO:39; Target sequence #6, SEQ ID NO:40 and SEQ ID NO:41; Target sequence #7, SEQ ID N042 and SEQ ID NO:43; and Target sequence #8, SEQ ID NO: 4 and SEQ ID NO:45.
  • Figure 14B shows two oligonucleotides of the present invention which can form triplexes with Target sequence #3: SEQ ID NO:46 and SEQ ID NO:47.
  • Figure 15A shows oligonucleotides containing unusual bases for use in triplex formation: II, SEQ ID NO:49; 12, SEQ ID NO:50; Gl, SEQ ID N0:51; and Al, SEQ ID NO:52.
  • Figure 15B presents two exemplary target sequences for oligonucleotide II (SEQ ID NO:48 and SEQ ID NO:53).
  • Potassium permanganate is sensitive to the secondary structure of nucleic acids, and it specif ⁇ ically reacts with exposed thyraines at the 5-6 double bond (Kochetkov & Budovskii, 1972) .
  • thymines in a single-stranded DNA are sensitive to KMn0 4 modification, but become resistant when in ⁇ volved in a duplex (Hayatsu & Ukita, 1967) .
  • Haner & Dervan (1991) have demonstrated that thymines on a single strand become less reactive toward KMn0 4 when the single strand is incorporated into a triple helix by occupying the major groove of a DNA duplex.
  • DMS reacts predominantly at the N-7 position of guanines (Maxam & Gilbert, 1980) .
  • the N-7 position of guanines in the Watson-Crick duplex is involved in hydrogen bonding to the third strand in both C + -GC and GGC base triplets; in the case of C + -GC triplets, guanines have been shown to become resistant to DMS modifica- tion (Johnston, 1988; Hanvey et al., 1988).
  • DEPC which carbethoxylates purines (A > G) primarily at the N-7 position, is useful for probing adenine residues involved in triplex formation.
  • adenines in the Watson-Crick strand show protection from DEPC modification because of the Hoogsteen hydrogen bonding involving the N-7 position (Johnston, 1988; Htun & Dahlberg, 1988; Hanvey et al., 1988). Similar protection is expected for the Watson-Crick adenine of the A-AT base triplet.
  • Oligonucleotide I ( Figure 8) has the general form (Pu) n N 4 (Py) n N 4 (Py) n and is expected to form an intramolecular triple helix with PyPuPy base trip ⁇ lets.
  • oligonucleotide II ( Figure 8) has the general form of (Py) n N 4 (Pu) n N 4 (Pu) n and thus has a potential of forming an intramolecular triplex with Pu-PuPy base triplets.
  • Oligonucleotides III-V consist of two different triplex-forming motifs fused together, i.e., these oligonucleotides can be considered as hybrids of oligonucleotides I and II. They belong to the general form (Pu) n (Py) n N 4 (Pu) ⁇ t (Py) n N 4 (Py) n (Pu) n and can potentially form a triplex consisting of a block of PyPuPy base triplets connected to a block of Pu.PuPy base triplets. For clarity, each oligo ⁇ nucleotide is described in three segments: 5', middle, and 3' segments.
  • the first two thymines at the 5' end remain hyperreactive toward KMn0 4 throughout all conditions, indicating that these bases are unpaired possibly because of "fraying," i.e., breathing or transient un-pairing of normally paired bases at an end of a molecule.
  • oligonucleo ⁇ tide II has the potential to form an intramolecular triplex with A-AT base triplets.
  • the reactivity patterns of oligonucleotide II in 50 mM sodium cacodylate (pH 7.1) buffer ( Figure 3, Example IB) support a triplex conformation.
  • the first four thymines (T j -T 4 ) at the 5' end are also reactive toward KMn0 4 ( Figure 3, lanes 2-4, arrows), suggesting that these bases are at least transiently unpaired or frayed.
  • intermolecular duplex or triplex pairing schemes for either oligonucleotides I or II that were consistent with the observed reactivities were unsuccessful.
  • an inter olecular duplex structure for oligonucleotide I should result in protection of thymines next to the adenine tract (T 9 -T 16 ) , not hyperreactivity as is observed.
  • Oligonucleotide III has two adjacent potential triple-helix forming motifs, one making T ⁇ T and the other making A-AT triplets. Thymines T 4 -T 7 on the 5' segment are unreactive toward KMn0 4 in 50 mM sodium cacodylate (pH 7.1) buffer (Example 1C; Figure 4A, lane 3) , suggesting that they are in a Watson-Crick duplex. In contrast to T 4 -T 7 , T ⁇ -T jg on the 3 r segment are modified by KMn0 4 ( Figure 4A, lane 3) , indicative of single-stranded character.
  • Oligonucleotide III was modified with DEPC in the same buffer to detect whether A ⁇ T triplets are formed, in addition to TAT triplets; the results are shown in lanes 9-12 of Figure 4A (Example 1C) .
  • Adenines 13-15 are protected from DEPC (open arrow ⁇ heads) , suggesting the formation of AAT base trip- lets.
  • the reactivity of these less-reactive adenines is not sensitive to the presence of Mg ++ ions. Because it is located in the loop region, the enhanced reactivity of A u (bottom closed arrowhead) is expected, and it serves as an internal control for the modification.
  • oligonucleotide IV also has the potential to form a triplex having juxtaposed Pu-PuPy and PyPuPy motifs, but with different base triplets (TAT and GGC) .
  • TAT and GGC base triplets
  • the results of KMn0 4 modification of oligonucleotide IV are shown in Figure 5A (Example ID) .
  • Thymines on the 3' segment (T ⁇ -T ⁇ ) are reactive toward KMh0 4 in TE buffer ( Figure 5A, lane 3, bracket) , indicating that they are in a single-stranded form.
  • T ⁇ -T ⁇ become less reactive ( Figure 5A, lanes 4-6) , suggesting the formation of T-AT base triplets.
  • T 17 -T 20 remain less reactive, consistent with their involvement in a duplex.
  • the reactivities of T 9 -T 12 and T ⁇ -T ⁇ do not change under any condition, indicating their singlestranded nature in the folded ( Figure 5A, lane 6) as well as unfolded structure ( Figure 5A, lane 3) .
  • oligonucleotide V consist of mixed purines and mixed pyrimidines. Unlike the case for other oligonucleo- tides, the formation of an intramolecular triplex by oligonucleotide V is expected to be pH-dependent because of the involvement of C + -GC base triplets.
  • Figure 6A Example IE.
  • Figure 6A Example IE
  • pH 4.5 both T 26 and T 28 on the 3' segment are protected from KMn0 4 modification ( Figure 6A, lane 3, arrowheads) whereas at pH 6 and 7 these two thymines are modified.
  • A-AT and GGC base triplets in oligonucleotide V is detected by reacting with DEPC and DMS, respectively (Example IE) .
  • DEPC open arrowheads
  • a ⁇ closed arrowhead
  • Both A j and A 4 are less reactive, consistent with their involvement in TAT base triplets.
  • Figure 6C shows the melting of oligonucleotide V as monitored by DEPC reactivity.
  • Figure 6C compare lanes 3-6 with lanes 7-10 (arrow heads)) .
  • G 3 and A 4 show a transition to increased reactivity well below 70'C even in the presence of Mg ++ ions, indicating the melting of C + -GC and T-AT base triplets. Therefore, in the presence of Mg ++ ions, A-AT base triplets in this mixed purine tract appear to be more stable than T-AT base triplets.
  • the different melting temperatures observed for A ⁇ T base triplets generat ⁇ ed within oligonucleotides V and III could be due to a sequence effect (oligonucleotide V contains 50% GC) .
  • the results of chemical modifica ⁇ tions of oligonucleotide V show the formation of intramolecular triplexes utilizing alternate strands of duplex DNA containing all four bases.
  • Experiments performed in support of the present invention show that using two types of base trip ⁇ lets, Pu-PuPy and PyPuPy, a single strand consisting of oligopurine- and oligopyrimidine-blocks bind simultaneously to a duplex DNA, where the duplex contains adjacent tracts of oligopurines and oligo- pyrimidines. This binding is via both Pu-PuPy and PyPuPy base triplets, as exemplified in Figure la(iii) .
  • the oligopyrimidine block of the single strand is parallel to one purine tract of the duplex DNA and its oligopurine block is anti- parallel to the other purine tract on the alternate strand of the duplex.
  • the present invention requires no special junction for this polarity of binding between the oligopurine and oligopyrimidine blocks of the single strand occupying the major groove of both oligopurine and oligopyrimidine tracts.
  • Oligonucleotides III-V containing tandem tracts of oligopurines and oligopyrimidines in different base combinations, exhibit chemical reactivity patterns that are in agreement with an intramolecular triplex structure forming both PuPuPy and PyPuPy base triplets simultaneously.
  • the reactivity patterns described above are consistent with the bases of a single-stranded region occupying the major groove of the duplex hairpin, base-pairing to the purine tract in each Watson-Crick strand, as depicted in Figure 1 (A and B).
  • the results of chemical modification performed at different temperatures suggest that Mg ++ ions enhance the stability of PuPuPy base triplets, although their formation does not require Mg ++ .
  • the third strand oligonucleotides of the present invention can be used in a number of diag- nostic and therapeutic applications.
  • One diagnostic application of the oligonucleotides of the present invention is to provide a means of site-directed cleavage at a target duplex by coupling oligonucleo ⁇ tides to cleaving agents.
  • One therapeutic applica- tion of the present invention is targeting a select ⁇ ed gene for inactivation, e.g., by suppression of mRNA synthesis from the target gene (Cooney et al., 1989; Orson et al., 1991; Postel et al. , 1991).
  • a defined oligonucle- otide is directed at the duplex DNA target to form a triple helix.
  • triple-helix approach for targeting DNA has had limited use to date because of the requirement for long homopurine target sequences.
  • triplex formation at an oligo ⁇ purine*oligopyrimidine typically required a single strand consisting of only either pyrimidines or only purines; the ability to use alternating tracts of oligopurine and oligopyrimidine sequences, as described above, obviates this requirement.
  • Oligonucleotides of the present invention are tested for triplex formation with duplex target sequences using band shift assays (Example 2) .
  • band shift assays Example 2
  • high percentage polyacrylamide gels are used for band-shift analysis where denaturing conditions (Ausubel et al. ; Sauer et al.; Sambrook et al.) are adjusted to reduce any background of non-specific binding.
  • a target duplex oligonucleotide is used initially for band shift analysis.
  • the duplex target is radiolabeled and mixed with a third strand oligonucleotide which is being tested for its ability to form triplex structures with the target duplex.
  • Oligonucleotides are evaluated for their ability to shift the mobility of the duplex oligonu ⁇ cleotide.
  • Triplex formation is indicated in the band shift assay by a decreased mobility in the gel of the labeled triplex structure relative to the labeled duplex structure, i.e., triplexes appear as bands having apparent higher molecular weight.
  • a number of controls are performed to assure the oligonucleotide specificity of triplex-forma ⁇ tion.
  • oligonucleotides having similar purine and pyrimidine compositions to the third strand oligonucleotide, but which have a random mix of the purine and pyrimidines instead of to the sequence specified by the duplex target are prepared. The random sequence oligonucleotides should neither affect the mobility of labeled duplex target nor interfere with the band-shift caused by sequence-specific third-strand oligonucleotide.
  • the advantage of using a chemical cleaving moiety is that, in addition to binding, the reagent permanently inactivates the target nucleic acid sequences.
  • This method is applicable to any double strand DNA target, and can be also be applied to selected target RNA molecules (see below) .
  • the present invention is particularly useful in target ⁇ ing viral genomes (free replicating or integrated) for inactivation.
  • HIV human immunodeficiency viruses
  • oligonucleotides are tested for triplex formation as described above.
  • the sequences of the oligonucleotides are chosen based on target sequences having tracts of oligo- purines-oligopyrimidines.
  • Figure 9A-9C illustrate three potential sites for targeting within the LTR region of HIV-1 with single-stranded oligonucleotides.
  • Figure 10 shows the sequences of oligonucleotides A 1 (SEQ ID NO:9) and 2 (SEQ ID NO:10), B 1 (SEQ ID NO:ll) and 2 (SEQ ID NO:12), and C 1 (SEQ ID NO:13) and 2 (SEQ ID NO:14) designed to target sequences of Figure 9A, 9B, and 9C, respec ⁇ tively.
  • Next to each of these oligonucleotides ( Figure 10) is the general pattern of base triplets expected to form when triplexes are formed.
  • Oligo ⁇ nucleotides K (SEQ ID NO:15), L (SEQ ID NO:16), and M (SEQ ID NO:17) are the control oligonucleotides ( Figure 10) .
  • All three target sites are located in the control region of the LTR, i.e., upstream of the transcription initiation site and therefore do not interact with mRNA sequences to function as anti ⁇ sense mediators.
  • the potential target sites A, B, and C ( Figure 9) have different triplex forming motifs: Site A, consisting exclusively of purines is targeted with oligonucleotides A-l and A-2 ( Figure 10) , which are capable of forming triplexes with Pu*PuPy and Py»PuPy base triplets, respective ⁇ ly.
  • Site B consists of a tract of pyrimidine residues flanked by two purine tracts and is target ⁇ ed with oligonucleotides B-l and B-2 ( Figure 10) , which have the correct polarities for triplex forma ⁇ tion.
  • Site C has some pyrimidines buried in a highly purine-rich sequence, and oligonucleotides C-1 and C-2 ( Figure 10) is directed toward site C.
  • Oligonu ⁇ cleotides K, L, and M each having incorrect polari- ty for triplex formation at sites A, B, and C, respectively, are used as controls. Test oligomers with and without phenanthroline are used to assess the effect of cleavage.
  • cleaving agents may be used in the present invention including the following chemical moieties which are capable of cleaving nucleic acid substrates: phenanthroline (Chen et al., 1986, 1987; Francois et al. , 1989; Ebright et al. , 1990), Fe(II)-EDTA (Dreyer et al. , 1985; Dervan, 1986; Moser et al. , 1987; Maher et al. , 1989; Sluka et al., 1987), Cu(II)-bipyridine, Cu(II)-terpyridine, and Zn(II) (Modak et al.
  • the cleaving agent 1,10-phenanthroline is attached to the third strand oligonucleotides as described in Example 3.
  • test oligonucleotides to form triplexes and carry out cleavage at their designated target sites is assayed by determining the site-specific cleavage induced at the target se ⁇ quences by test oligonucleotides equipped with the phenanthroline moiety (Example 4) .
  • pHIV-lCAT is linear ⁇ ized with, end-labeled, and subjected to a second restriction digest to obtain a uniquely labeled DNA fragment. After gel purification, this DNA fragment is mixed with a phenanthroline modified oligonucleo- tide. Cleavage products are resolved on sequencing gels along with the products of sequencing reac ⁇ tions.
  • This method maps the site of triplex forma ⁇ tion, and provides an indication of the cleavage efficiency (detected by counting the radioactivity of excised gel bands) ; the method allows the quantitation of the efficiency of triplex formation.
  • In vitro triplex formation can also be tested using the band shift assay described above, where cleavage of the duplex target is evidenced by increased mobility of the fragments of the labeled duplexes in the gel.
  • polyacryl- amide gels run under DNA-denaturing conditions (Ausubel et al.; Sambrook et al.) can be used.
  • phenanthroline- coupled-oligonucleotides To examine the ability of the phenanthroline- coupled-oligonucleotides to inhibit gene expression by in vivo triplex formation, transient expression of the CAT gene under the direction of HIV-1 LTR in HeLa cells is used.
  • HeLa cells are transfected with pHIV-1 CAT, using the DEAE-dextran technique (Queen and Baltimore, 1983) or Ca 3 (P0 4 ) 2 technique.
  • Oligo- nucleotides carrying phenanthroline are complexed with CuS0 4 before they are introduced to the cell medium. Approximately twelve hours after transfec- tion, the cells are incubated with a phenathroline- coupled-oligonucleotide, as described by Postel et al. (1991) .
  • Mercaptopropionic acid or ascorbic acid are then supplied to the medium approximately 12 hr after the oligonucleotide treatment.
  • Mitomycin C is added to the medium to induce CAT expression; since the HIV-1 LTR is under the influence of NF-kB, the expression of CAT activity can be induced by treating with either ultraviolet light or mitomycin C.
  • Cells are harvested at 12 and 24 hr after mitomycin C addition, and CAT activities determined as described by Gorman et al. (1982) .
  • CAT expression is compared to controls including cells that have been exposed to control phenanthro- line-coupled-oligonucleotides (K, L, and M) , phenan- throline-coupled-oligonucleotides (SEQ ID N0:9-SEQ ID NO:17) without complexing to CuS0 4 , and cells without oligonucleotide treatment.
  • Successful cleavage of the target by the duplex-targeted third strand oligonucleotide reduces levels of CAT expres- sion relative to controls, in particular, relative to phenanthroline-coupled-oligonucleotides (K, L, and M) complexed to CuS0 4 .
  • HIV-reporter gene constructs can be used in the above experiments (e.g., Cross et al.), including HIV-LTR constructs containing a luciferase (de Wet et al.) reporter gene.
  • oligonucleotide Inhibition In addition to the above-described site direct ⁇ ed cleavage of target DNA duplexes by the oligonu ⁇ cleotides of the present invention, oligonucleotides can be constructed to target duplex DNA regions in order to block expression — without cleavage. Target sites can be chosen in the control region of the genes, e.g., in the transcription initiation site or binding regions of regulatory proteins (Helene and Toulme, 1990; Birg et al., 1990; Postel et al. , 1991; Cooney et al. , 1988). These oligonucleotides can also interact with mRNA sequences to function as antisense mediators (see below) .
  • Single-stranded DNA can also be used as a target nucleic acid for oligonucleotides of the present invention.
  • two oligonucleo ⁇ tides can be used for target-directed binding.
  • oligonucleotides similar to those described below (TDAs) can be used for the binding or binding/cleavage of target sequences contained in single-stranded DNA molecules.
  • Single-stranded oligonucleotides of the present invention can also be used in an anti-sense expres ⁇ sion inhibition technique, where the single-stranded oligonucleotides are capable of forming triplex structures with target m NAs (Giovannangeli et al., 1991) .
  • This triplex-directed antisense (TDA) approach may be more effective than a duplex (con ⁇ ventional antisense approach) in arresting biologi ⁇ cal processes such as translation and reverse transcription.
  • Triplex formation in this fashion is highly selective and of high affinity and may not be a substrate for enzymes such as helicases. The action of such helicases can be a potential problem in the conventional antisense approach.
  • TDA approach of the present invention is applicable to any target mRNA molecule; an example of targeting HIV mRNA is presented below.
  • Three TDA target sites (D, E, and F) within the 5786-8887 nucleotide region of the HIV-l genome are presented in Figure 12A.
  • the figure also shows a target region for each mRNA sequence (underlined) .
  • TDA oligonucleotides directed to these sequences are presented as oligonucleotides Dl, D2, El, E2, and Fl, F2 in Figure 12B.
  • Oligonucleotides complementa ⁇ ry to the target regions i.e., oligonucleotides designed to function as conventional antisense agents, are presented as oligonucleotides D x , E x , and F x in Figure 12B.
  • oligonucleotides capable of forming triplexes are synthesized. Two different triplexes, one with Pu»PuPy and the other with Py»PuPy base triplets, are possible for each target ( Figure 12B) .
  • connecting sequences are synthesized with the "UNI-LINK AMINO- MODIFIER" (Clonetech, Palo Alto CA) , which lacks a base and a sugar attached to the phosphate.
  • TDA oligonucleotides are assayed by detecting the expression of gpl20 (enve ⁇ lope glycoprotein) of HIV.
  • HeLa T4+ cells AIDS Research and Reference Program
  • pDOLHIVenv available from the AIDS Research and Reference Program
  • the pDOLHIVenv plasmid has open reading frames for Env, Tat, and Rev proteins (nucleotides 5786-8887 of the HIV-1 genome) .
  • j ⁇ -Kanamycin-resistant cells are picked and expanded. These cells are then incubated with a selected TDA oligonucleotide for 24 hr.
  • the level of expression of envelope proteins is assayed by a syncytium (giant cell) formation assay carried out in microtiter plates (Putney, et al. , 1989). Because syncytium induction by HIV is mediated by the interaction of the CD4 molecule with gpl20 on the surface of cells, the number of syncytia is directly proportional to the amount of expressed gpl20.
  • oligonucleotides D2, E2, and F2 which are capable of direct binding to mRNA, are used as controls in gpl20 expression experiments.
  • an indirect immunofluorescence assay is employed to detect the level of gpl20 expression.
  • an antiserum to gpl20 RF available from the AIDS Research and Reference Program
  • Rhodamine isothiocya- nate-conjugated rabbit anti-goat immunoglobulin G (Pierce, Rockford, IL) is used as the secondary detection antibody.
  • TDA oligonucleotides can be targeted to regula ⁇ tory regions, as described above, or to other regions, such as splice sites in pre-mRNA molecules.
  • Triple helix formation is extremely sequence- specific and bases that can form hydrogen bonds with Watson-Crick base pairs can form triplexes. Howev ⁇ er, the relative stabilities which may vary from one base triplex to the other is important in designing single strand probes for triplex formation.
  • the affinity cleaving assay described above is used to detect the relative propensity of triplex formation involving oligonucleotides containing the above unusual base triplets.
  • the propensity of triplex forma ⁇ tion by I*GC with respect to that of G»GC is assayed by comparing the cleavage efficiency induced by oli ⁇ gonucleotide II and Gl ( Figure 15B) .
  • the polarity of the third strand as well as the type of neighboring base triplets would be the same except for variants, i.e., I»GC and G»GC base triplets.
  • the difference of cleavage efficien ⁇ cy induced by these two oligonucleotides simply reflect the propensity of triplex formation by I»GC and G»GC base triplets.
  • the propensity of the formation of I»AT triplet with respect to that of A «AT is be assayed using oligonucleotide 12 and Al ( Figure 15B) .
  • oligonucleotide II will be used to detect targeting in the duplex sequences shown in
  • the oligonucleotides of the present invention comprise tandem, alternating tracts of purine and pyrimidine sequences, where the length of each tract may vary but contains at least one purine or pyrimi ⁇ dine base (Example 4B) .
  • the tracts of purines and pyrimidines contain at least 4 purine bases followed by at least 4 pyrimidine bases.
  • the number of bases in the tracts of purines and pyrimi ⁇ dines do not have to be equal, for example, a tract of 4 purines may be attached to a tract of 9 pyrimi ⁇ dines, or the oligonucleotide may comprise tandem tracts of 4 purines, 9 pyrimidines, 2 purines, 5 pyrimidines.
  • the typical length of targets for the oligonucleotides of the present invention are from about 8 nucleotides to about 60 nucleotides; the length of the target is used, in part, to determine target specificity and triplex stability.
  • Cellular uptake of the 32 P-labeled oligonucleo ⁇ tides is evaluated essentially as described by Postel et al.
  • Stability of the oligonucleo ⁇ tides in vivo can be evaluated by freeze-drying aliquots from the medium and from the nuclear and cytoplasmic fractions and analyzing the resuspended aliquots on 12% sequencing gels (Postel et al.).
  • oligonucleotides of the present invention can be facilitated using established techniques such as scrape loading, electroporation, and microinjection.
  • oligonucleotides can be encapsulated within lipo ⁇ somes and these loaded liposomes incubated with cells.
  • oligonucleotides can be protected by attaching intercalators; this has been shown to make oligonucleotides resistant to exonucleases and facilitates cell permeation (Cazenave et al., 1987) .
  • the linking of intercalators improves the exonucle- ase resistance of oligonucleotides and further provides additional binding energy (because of intercalation) without perturbing the specificity of recognition of complementary sequences.
  • Backbone modifications such as methylphosphonates and phos- phothioates not only make oligonucleotides endonu- clease resistant, but the neutral backbones also facilitate cell permeation (Matteucci et al.
  • Oligonucleotides at the 5' and 3' ends can be derivatized with two different linking molecules using different chemistry. Such derivatization is performed by using "3'-AMINE-ON CPG" (Clontech) solid supports to incorporate a functional amino group at the 3' end, and incorporating a thiol group at the 5' end as described above. Because deprotec- tion procedures for the two functional groups are different and independent from each other, derivati ⁇ zation at the two ends is performed at two stages of oligonucleotide synthesis.
  • TDA oligonucleotides can be derivatized at one or both ends to modify the oligonucleotides to provide a cleavage function, as described above; RNA targets can be cleaved using the cleaving moieties described above, including 1,10 phenanthro ⁇ line.
  • the intracellular reduction potential can be modulated using N-acetyl cysteine, which increas ⁇ es the intracellular glutathione level (Roederer et al. , 1990; Kalebic et al. , 1991).
  • Such manipulation of the intracellular reduction should assist in keeping, for example, a copper atom of a cleaving agent in the reduced state.
  • the level of expression of cytoplasmic mRNA corresponding to target sequences can be evaluated by standard methods including: Northern analysis using a target mRNA specific probe (Ausubel et al. ; Sambrook et al.; Postel et al.) ; dot-blot hybridiza ⁇ tions using target mRNA specific probes (Ausubel et al.); and polymerase chain reaction (Mullis; Mullis et al.) using target mRNA specific probes.
  • substitution of Br5U (5- bromo-uracil) for thymines in the third strand increases binding affinity when a triplex is formed. Therefore, thymines that form Hoogstein hydrogen bonds can be replaced with Br5U during chemical synthesis. Further, the substitution of Me5C (5- methyl-cytosine) in place of cytosines in the third strand is useful for effective targeting at physio ⁇ logical pH.
  • oligonucleotides of the present invention to recognize targets having combinations of tracts of purines and pyrimidines provides flexibility for techniques based on triplex formation, including third strand inactivation of a target duplex gene and anti-sense inactivation (Helene and Toul e, 1990) .
  • cleaving reagents of the present invention provide means for a method of cleaving RNA targets at specific sites. Such cleavage is useful for the analysis of RNA structure and function as well as diagnostic analyses.
  • a diagnostic application is to isolate RNA from a cell infected with a particular RNA virus. Total or poly-A+ RNA (Ausubel et al.) is end labeled. The RNA is then isolated away from free label and the amount of incorporated label estimated, for example, by scintillation counting.
  • RNA cleaving agent such as a TDA oligonucleotide combined with a chemical cleaving moiety
  • the amount of liberated label is used as an indicator of the concentration of RNA contain the RNA-binding protein cognate binding site.
  • the cleaving reagents of the present invention are particularly desirable for use with DNA virus targets and RNA virus targets or their pro-viral DNA forms: for example, cleaving HIV genomic RNA or pro-viral DNA.
  • Oligonucleotides of the present invention capable of forming triple-helix structures with single-strand or duplex DNA, can also be labeled and used as probes, or, when carrying cleaving agents, can be used as site-specific cleaving agents.
  • the cleaving reagents of the present invention are also useful in a method of inhibiting expression of RNA viral (e.g., HIV) antigens in cells infected with the virus.
  • infected cells are exposed to an oligonucleotide (DNA duplex directed, antisense, or TDA) modified to contain a cleaving moiety (i.e., the reagent), at a reagent concentration effective to produce reduction in viral antigen expression in the infected cells (Wang et al., 1988, 1989; Crowe et al., 1990).
  • oligonucleotide DNA duplex directed, antisense, or TDA
  • TDA cleaving moiety
  • a combined use of RNA cleaving (or inhibiting) oligonucleotides combined with the above-described DNA duplex directed cleaving (or inhibiting) oligo ⁇ nucleotides may provide a two-pronged therapeutic attack against viral diseases by providing cleavage of viral RNA and DNA or pro-viral DNA genomes.
  • a major advantage of targeting the DNA pro-virus associated with an RNA virus is that typically only one, or a few copies, of integrated, transcriptio- nally active DNA are present per cell in contrast to many copies of mRNA which may be present in an infected cell (Soma et al. , 1988) .
  • Oligonucleotides were synthesized on an Applied Biosystems Model 381 B automated DNA synthesizer (Applied Biosystems, Foster City CA) using cyano- ethyl phosphoramidites. After deprotection, they were purified by electrophoresis on denaturing 20% polyacrylamide gels (Ausubel et al. ; Sambrook et al.) . Purified oligonucleotides were labeled at 5' ends using T 4 polynucleotide kinase (United States Biochemicals, Cleveland OH) and 32 P- ⁇ -ATP (Du Pont-NEN, Boston MA) according to the method of Maxam & Gilbert (1980) .
  • Unincorporated ATP was separated (Ausubel et al. ; Sambrook et al.) from labeled oligonucleotides by passing the labeling reaction mixture through two successive "SEPHADEX G-50" spin columns (Pharmacia, Piscataway NJ) equilibrated in 10 mM Tris-HCl and 0.1 mM EDTA (pH 7.5).
  • oligonucleotides usingpotassi ⁇ um permanganate were performed essentially as described by Rubin & Schimid (1980) .
  • a radiolabeled oligonucleotide (approximately 100 ng) was equili ⁇ brated in 9 ml. of the reaction buffer (see below) at 4'C for 10 min, then 1 ml. of 5 mM KMn0 4 (freshly diluted from a 100 mM stock stored at 4'C) was added. After 10 min of incubation at 4'C, the reaction was stopped by adding 2 ml. of neat allyl alcohol.
  • oligonucleotides using diethyl pyrocarbonate were performed essentially as described by Johnston & Rich (1985) or Herr (1985) .
  • An end-labeled oligonucleotide (approximately 100 ng) was equilibrated in 97.5 ml. of a reaction buffer (see below) for 10 min at 4°C.
  • 2.5 ml. of DEPC Aldrich, Milwaukee WI
  • oligonucleotides using dimethyl sulfate was performed essentially as described by Maxa & Gilbert (1980) .
  • One microliter of a 1:200 dilution of DMS in water (freshly prepared) was added to an end-labeled oligonucleotide (approx ⁇ imately 100 ng) equilibrated in 9 ml. of reaction buffer (see below) at 4'C, and the modification was carried out for 10 min at the same temperature.
  • DNA pellets were thoroughly washed with 70% ethanol, dried, and treated with hot piperidine (1 M, 90 * C for 30 min) .
  • KMn0 4 -modified DNA was treated with piperidine directly, without ethanol precipita ⁇ tion. After removing piperidine by vacuum evapora- tion, DNAs were resuspended in formamide loading buffer, and the cleavage products were resolved on 20% sequencing gels. Sequencing reactions on oligo ⁇ nucleotides were performed according to Williamson & Celander (1990) .
  • KMn0 4 reactivity of oligonucleotide I was performed in 10 mM Tris-HCl (pH 7.0) and 0.1 mM EDTA (TE) buffer at 4'C in the absence ( Figure 2, lane 2) and presence of MgCl 2 ( Figure 2, lanes 3-5, concentrations as indicated) .
  • Figure 2, lane 1 is the A+G sequencing reaction of oligonucleotide I (Maxam and Gilbert) . Modification of thymines within the bracket, " ⁇ " indicated in lane 1, is sensitive to MgCl 2 .
  • the open and closed bars, indicated on the right, represent WatsonCrick duplex region; single lines show single-stranded regions; and the hatched bar indicates the third "strand" of the triplex.
  • Open arrowheads show adenines (A 13 -A 16 ) that are protected from DEPC modification, whereas closed arrowheads represent reactive adenines (A ⁇ -A ⁇ ) .
  • Helical and loop regions are designated as described above for Figure 2.
  • lane 1 is the G sequencing reaction; lane 2, is the A+G sequencing reaction.
  • DMS reactivity as a function of tempera ⁇ ture DMS reactivity as a function of temperature was examined in the absence ( Figure 5B, lanes 1-5) and presence (Figure 5B, lanes 6-10) of 10 mM MgCl 2 . Temperatures were as indicated at the top of the figure. Bracketed bases (]) are affected by MgCl 2 concentration, indicated in Figure 5A, or tempera ⁇ ture, indicated in Figure 5B. Helical and loop regions are as described above for Figure 2.
  • Example 2 Band Shift Assays Triplex formation is analyzed essentially as described by Roberts et al. (1991) .
  • Figure 9A shows a sample target duplex sequence (SEQ ID NO:6). A representative target sequence to be used for triplex formation is shown in bold;
  • Figure lOA-1 and 10A-2 illustrate the sequences of sample third strand oligonucleotides (SEQ ID NO:9 and SEQ ID NO:10) .
  • the annealing reaction involves mixing the two purified oligonucleotides in IX TE+50 buffer (10 mM Tris HCl pH 7.4, 1 mM EDTA, 50 mM NaCl) , or other suitable buffer (Sauer et al.) .
  • the annealing mixture is heated to approximately 85°C and cooled slowly to room temperature.
  • This duplex DNA is then end-labeled using polynucleotide kinase and 7 ⁇ 32 P-ATP (Ausubel et al.) as described above.
  • the radiolabeled duplex is then mixed individu ⁇ ally with each third strand oligonucleotide being tested in 20 mM HEPES, pH 7.1, 50 mM NaCl, 5mM spermine.
  • the oligonucleotide single- strand is added in vast excess (e.g., 10 micromolar) to the 32 P-labeled duplex oligonucleotide (e.g., 1.0 nM) .
  • Reaction volumes are typically 15-20 microli- ters.
  • the samples are then loaded on a 20% acryla- mide non-denaturing gel, acrylamide/bisacrylamide 19:1, formed using 20 mM HEPES, pH 7.1 buffer.
  • the running buffer of the gels is (same hepes 20 mM , 7.1.
  • the gels are typically run at 6V/cm at 4°C.
  • the gels are dried and exposed to X-ray film.
  • the formation of triplex complexes is detected by a band shift, by reduction in the intensity of the duplex band and the appearance of a higher molecular weight band, which contains the duplex complexed to the test oligonucleotide to form the triplex struc ⁇ ture.
  • the concentration of duplex can be held constant and the concentration of the test oligonucleotide varied over a concentration range to give an idea of concentration effects associated with the test oligonucleotide.
  • Any oli ⁇ gonucleotide which potentially can form triplex structures with a given target duplex, can be tested as described above.
  • each oligonucle- otide is synthesized with a thiol group at the 5' end using the "C6-THIOL MODIFIER" reagent from Clonetech (Palo Alto, CA) , according to the manufac ⁇ turers instructions. Briefly, the oligonucleotides are deprotected after synthesis with NH 4 OH and then treated with silver nitrate to expose the thiol group. The oligonucleotide is immediately reacted with 5-iodoacetamido 1,10-phenanthroline.
  • the resulting oligonucleotides are separated from un-reacted iodo compound by passing the reac ⁇ tion mixtures through "SEPHADEX G-50" spin columns (Pharmacia, Piscataway NJ) .
  • Phenanthroline attached to an oligonucleotide binds cupric ion and this complex can be used to cleave DNA.
  • the bound cupric ion is reduced to cuprous ion, which reduces molecular oxygen to produce hydrogen perox ⁇ ide.
  • the E ⁇ O j reacts with the cuprous complex to form a copperoxo species that is directly responsi- ble for cleavage (Sigman, 1990) .
  • pHIV-lLTR-CAT Substrate.
  • pHIV-lLTR-CAT S. Miller, SRI International, Menlo Park CA
  • Hindlll Boehrin- ger Mannheim, Indianapolis IN
  • NNN 32 P- ⁇ -ATP
  • polynucleotide kinase and subjected to a second restriction digest to obtain a uniquely labeled DNA fragment, i.e., one carrying label only at one end of the linearized molecule.
  • the pHIV-lLTR-CAT fragment carrying the target sequence and end-label is gel purified (Ausubel et al.; Sambrook et al.) .
  • this DNA fragment is mixed with phenanthroline modified oligonucleotide in a buffer containing 10 mM Tris-HCl, 100 mM NaCl, 500 mM spermine, and 20 mM MgCl 2 .
  • the pH of the buffer is adjusted depending on the sequence of the target (a lower pH is used for the formation of C + *GC base triplets).
  • cleavage is initiated by adding CuS0 4 (to 10 mM) and mercaptopropionic acid (to 2.5 mM) . Cleavage products are resolved on sequencing gels along with the products of sequencing reactions for the pHIV- 1LTR-CAT target fragment.
  • the oligonucleotide sequences ( Figure 14A) are inserted into pUC18 (Bethesda Research Laboratories, Gaithersburg MD) between the EcoRI and BamHI sites.
  • the recombinant plasmids are purified and used as substrates for affinity cleaving assay.
  • affinity cleaving assay single-stranded oligonucle ⁇ otides covalently bound to Cu(II)-1,10-phenanthro- line are used as third strands. Two different third strands are possible for each target sequence; both candidates are synthesized and studied.
  • the two oligonucleotides shown in Figure 14B can form triplexes with the duplex target sequence #3 shown in Figure 14A.
  • the cleavage reactions are carried out and analyzed essentially as described above.
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Abstract

An oligonucleotide capable of forming a triple-helix with a nucleic acid molecule containing a target nucleic acid sequence is described, where the oligonucleotide is comprised of tandem, alternating tracts of purine and pyrimidine sequences, and where the oligonucleotide is effective to form a triple-helix structure with a target nucleic acid sequence having alternating tracts of purine and pyrimidine sequences. A method of cleaving the nucleic acid molecule is also described.

Description

TRIPLE-HELIX FORMATION
AT rPUNPYN) • fPU PYN1 TRACTS
i- Field of the Invention
The present invention relates to the use of triple helical DNA in the control of gene expres-
10 sion, including therapeutics, and the development of sequence-specific DNA cleaving agents. The present invention describes third strand molecules that utilize both Pu*PuPy and Py«PuPy base triplets to form a continuous DNA triple-helix at tandem oligo-
15 purine (Pun) and oligopyri idine (Pyn) tracts.
References
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Zamecnik, P.C., et al. , Proc. Natl. Acad. Sci. USA 82, 4143-4146, (1986). Background of the Invention
Much recent research has been focussed on the nucleic acid triple helix, a structure that was discovered more than 30 years ago in synthetic poly- ribonucleotides consisting exclusively of purine and pyrimidine strands (Felsenfeld et al., 1957). Triplex DNA has been shown to form in (i) oligo- purine-oligopyrimidine mirrorrepeats under superhel- ical stress (H-DNA) (Lyamichev et al. , 1986; Mirkin et al., 1987; Voloshin et al. , 1988; Htun & Dahl- berg, 1988; Kohwi & Kohwi-Shigematsu, 1988; Hanvey et al. , 1988; Wells et al. , 1988; Johnston, 1988; Glover & Pulleyblank, 1990) , (ii) between linear singl strands and native duplex DNA (intermolecular triplexes) (Moser & Dervan, 1987; Francois et al., 1988; Pilch et al., 1990), and (iii) within linear single strands (intramolecular triplexes) (Haner & Dervan, 1990; Chen, 1991) under suitable conditions. Although a detailed X-ray crystal structure of a triple helix is still lacking, a substantial body of chemical and physical evidence indicates that recognition of a duplex oligopurine-oligopyrimidine tract by an oligopyrimidine single strand [Figure la(i) ] involves the occupation of the major groove of the duplex by the oligopyrimidine strand and the formation of the PyPuPy base triplets T-AT and C+"GC via Hoogsteen hydrogen bonding (Felsenfeld et al., 1957; Morgan & Wells, 1968; Lee et al., 1979; Praseuth et al., 1988; Johnston, 1988; Rajagopal & Feigon, 1989; de los Santos et al., 1989). The oligopyrimidine strand lies in parallel orientation to the oligopurine tract of the duplex (Moser & Dervan 1987; Praseuth et al., 1988) , and the pairing of T with AT and C+with GC makes the recognition sequence-specific. Because the C+"GC triplet re¬ quires protonated cytosines on the Hoogsteen (third) strand, triplex formation is facilitated by low pH (Mirkin et al., 1987).
In addition to PyPuPy triplets, another general class of base triplets, Pu-PuPy (GGC and A"AT) , has also been proposed to explain the sequence-specific recognition of oligopurineoligopyrimidine tracts in duplex DNA by oligopurine single strands (Figure la(ii)). In this case the oligopurine single strand, which again lies in the major groove, is antiparallel to the oligopurine tract in duplex DNA
(Beal & Dervan, 1991) . Triplexes of the Pu-PuPy type can form independently of pH (Kohwi &
Kohwi-Shigematsu, 1988; Kohwi-Shigematsu & Kohwi,
1991) and thus are potentially more useful in many applications than triplexes of the PyPuPy type.
In addition to the above base triplets, unusual triplets such as GTA (Griffin & Dervan, 1989; Radhakrishnan et al., 1991), C+(or C)-AT, and UGC (Pei et al., 1991) and unnatural triplets such as TAT and I-GC (Letai et al., 1988) have also been documented in triple helical nucleic acids.
Typically, DNA recognition via triplex forma¬ tion has been restricted to oligopurineoligopyri i- dine tracts using single strands consisting exclu- sively of either purines or pyrimidines. The following approaches (Home & Dervan, 1990) have been proposed for recognition of all four bases in triplex formation: (1) synthesizing novel, unnatu¬ ral bases to complete the triplet code; (2) exclud- ing the recognition of certain base pairs in a triplex motif by incorporating abasic sites in the single strand, which may decrease the specificity of triplex formation; and (3) designing homopyrimidine single strands capable of binding to alternate strands of duplex DNA. In the last approach, polarity considerations required the design of an oligonucleotide with an unnatural 3 '-3 ' linkage having the proper spacing between two oligopyrimi¬ dine strings (Home & Dervan, 1990) .
Experiments performed in support of the present invention suggest a different approach to achieve recognition of all four bases in triplex formation.
This approach does not require the synthesis of unusual bases or linkages.
Summary of the Invention An oligonucleotide capable of forming a triple- helix with a nucleic acid molecule containing a target nucleic acid sequence is described, where the oligonucleotide is comprised of tandem, alternating tracts of purine and pyrimidine sequences, and where the oligonucleotide is effective to form a triple- helix structure with a target nucleic acid sequence having alternating tracts of purine and pyrimidine sequences.
The oligonucleotide of the present invention typically contain at least one purine tract adjacent at least one pyrimidine tract. Also, the target sequence, which oligonucleotide binding is directed toward, usually contains at least about 8 nucleo- tides. The nucleic acid target sequences of the present invention can be duplex DNA molecules, single-strand DNA molecules, and/or RNA molecules. Typically, when the nucleic acid molecule is a single stranded nucleic acid molecule, and the oli- gonucleotide contributes two of the three strands of the triple-helix.
In one embodiment of the present invention, the oligonucleotide further includes at least one moiety attached to the oligonucleotide, where the moiety is capable of cleaving the DNA molecule containing the target nucleic acid sequence. A representative group of such cleaving moieties includes the follow- ing: phenanthroline Cu(II) , Zn(II), Fe(II)-EDTA, Cu(II)-bipyridine, and Cu(II)-terpyridine.
Also described is a method of cleaving a nucleic acid molecule containing a target nucleic acid sequence, wherein the nucleic acid molecule is contacted with an oligonucleotide comprised of tandem, alternating tracts of purine and pyrimidine sequences, where the oligonucleotide is effective to form a triple-helix structure with a target nucleic acid sequence having alternating tracts of purine and pyrimidine sequences, and where the oligonucleo¬ tide includes at least one moiety which is capable of cleaving the nucleic acid molecule containing the target nucleic acid sequence. In this method, the oligonucleotide concentration is effective to produce cleavage of the nucleic acid molecule containing the target nucleic acid sequence
The target nucleic acid can be single- or double- stranded; this includes DNA and RNA targets.
Brief Description of the Figures
Figure 1. A. Schematic illustration of different triplex motifs, (i) A PyPuPy triple helix in which the third, oligopyrimidine strand is parallel to the purine strand of the duplex. (ii) A PuPuPy triple helix in which the third, oligopu¬ rine strand is antiparallel to the purine strand of the duplex. (iii) A triplex spanning tandem oligo¬ purine and oligopyrimidine tracts in which the purine block of the third strand forms hydrogen bonds with purines of one strand of the duplex (via Pu-PuPy base triplets) , whereas the pyrimidine block of the third strand hydrogen bonds to the purine tract of the other strand of the duplex via PyPuPy base triplets. Within this hybrid triplex, the required polarity of the third strand is maintained. The polarity of each strand is shown by a half-ar- row. Open circles represent Watson-Crick hydrogen bonding and closed circles represent Hoogsteen or another type of hydrogen bonding.
B. A ribbon model for an intramolecular triplex of type iii, in which the third strand recognizes alternate strands of a hairpin duplex. Here the third-strand region (shown in the middle in gray and black-and-white stripes) folds back on the hairpin duplex (white ribbon) , forming two loops (top and bottom) . The purine tract of the third strand (gray) forms base pairs (black bars) with the purine tract of one strand of the Watson-Crick hairpin (making Pu-PuPy base triplets) , whereas the pyrimidine tract of the third strand (black-and— white) forms Hoogsteen base pairs (banded bars) with the purine tract of the other Watson-Crick strand making PyPuPy base triplets. White bars represent Watson-Crick hydrogen bonding, and arrows indicate the polarity (5' to 3') of the strand. Figure 2. This figure illustrates the KMn04 reactivity of oligonucleotide I (SEQ ID N0:1) (Figure 8) .
Figure 3. This figure illustrates the KMn04 Λni DEpcreactivity of oligonucleotide II (SEQ ID NO:2) (Figure 8) .
Figure 4. This figure illustrates the KMn04 Md DEPCreactivity of oligonucleotide III (SEQ ID NO:3) (Figure 8) .
Figure 5. This figure illustrates the KMn04 tnd DEPCreactivity of oligonucleotide IV (SEQ ID NO:4) (Figure 8) .
Figure 6. This figure illustrates the KMn04 DEPc. and DMSreactivity of oligonucleotide V (SEQ ID NO:5) (Figure 8) . Figure 7. This figure summarizes the reactivi¬ ty patterns and deduced secondary structures for each of oligonucleotides I-V: Closed arrows, KMn04 reactivity that is not sensitive to Mg++ ions; the size of the arrow roughly correlates with the extent of base reactivity. (The arrow shown in parentheses is a presumably reactive thymine that was run off the gel.) Open arrows, KMn04 reactivity of thymines sensitive to the presence of Mg++ions. Open rectan¬ gles, DEPC reactivity of adenines, with the symbol size roughly correlating with the extent of reactiv¬ ity. Open cigar-shaped symbols, DMS reactivity of guanines. Lines connecting bases show the continu¬ ity of the backbone. Open circles, Watson-Crick hydrogen bonding. Closed circles, Hoogsteen or PuPu-type hydrogen bonding.
Figure 8. This figure presents the nucleotide sequences of oligonucleotides I-V.
Figure 9 (A-C) illustrates three potential sites for targeting within the LTR region of HIV-1 with single-stranded oligonucleotides.
Figure 10 presents the sequences of oligonucle- otides Al (SEQ ID NO:9) and A2 (SEQ ID N0:10), Bl (SEQ ID NO-.ll) and B2 (SEQ ID NO:12), Cl (SEQ ID NO:13) and C2 (SEQ ID NO:14) which are designed to target sequences of Figure 9A, 9B, and 9C, respec¬ tively. Next to each of these oligonucleotides is the general pattern of base triplets expected to form when triplexes are formed. Oligonucleotides K (SEQ ID NO:15), L (SEQ ID NO:16), and M (SEQ ID NO:17) are oligonucleotides used in control experi¬ ments. Figure 11 illustrates the chemistry of the attachment of a phenanthroline moiety to a cysteine- containing polypeptide.
Figure 12A illustrates three TDA target sites (D, SEQ ID NO:18; E, SEQ ID NO:19; F, SEQ ID NO:20) within the 5786-8887 nucleotide region of the HIV-1 genome. Figure 12B shows a schematic representation of triplex formation at target mRNA sequences D, E, and F. For each mRNA sequence two oligonucleotides
(1 and 2) are shown: Dl (SEQ ID NO:21), D2 (SEQ ID
NO:22); El (SEQ ID NO:23), E2 (SEQ ID NO:24); and Fl
(SEQ ID NO : 25) , F2 (SEQ ID NO : 26) . Oligonucleotides Dχ (SEQ ID NO : 27) , Eχ (SEQ ID NO: 28) , and Fχ (SEQ ID
NO:29) are the complementary oligonucleotides designed to function as conventional antisense agents. X indicate abasic sites of oligonucleotides
— in the above listed SEQ ID NOs., the abasic site is indicated by an N.
Figure 13 provides an overview of a method for targeting and inactivation of HIV mRNA using TDA oligonucleotides.
Figure 14A shows oligonucleotide sequences containing the target sequences for Example 4B. These sequences are also presented as: Target sequence #1, SEQ ID NO:30 and SEQ ID NO: 31; Target sequence #2, SEQ ID NO:32 and SEQ ID NO:33; Target sequence #3, SEQ ID NO:34 and SEQ ID NO:35; Target sequence #4, SEQ ID NO:36 and SEQ ID NO:37; Target sequence #5, SEQ ID NO;38 and SEQ ID NO:39; Target sequence #6, SEQ ID NO:40 and SEQ ID NO:41; Target sequence #7, SEQ ID N042 and SEQ ID NO:43; and Target sequence #8, SEQ ID NO: 4 and SEQ ID NO:45. Figure 14B shows two oligonucleotides of the present invention which can form triplexes with Target sequence #3: SEQ ID NO:46 and SEQ ID NO:47.
Figure 15A shows oligonucleotides containing unusual bases for use in triplex formation: II, SEQ ID NO:49; 12, SEQ ID NO:50; Gl, SEQ ID N0:51; and Al, SEQ ID NO:52. Figure 15B presents two exemplary target sequences for oligonucleotide II (SEQ ID NO:48 and SEQ ID NO:53). Detailed Description of the Invention
I. Intramolecular Triple-Helix Formation. Oligonucleotides were synthesized which were designed to fold into intramolecular triplexes as shown schematically in Figure 7. Analysis of these oligonucleotides using several different chemical probes indicated that triple-helix formation occurs at tandem oligopurine and oligopyrimidine tracts. This finding suggests that designing oligonucleo- tides for triplex binding, so as to utilize both known types of base triplets together with strand switching, can significantly relax the requirement of homopurine sequences for triplex formation.
The following chemical reagents have been used in the analysis of triplex formation using the oli¬ gonucleotides of the present invention.
Potassium permanganate is sensitive to the secondary structure of nucleic acids, and it specif¬ ically reacts with exposed thyraines at the 5-6 double bond (Kochetkov & Budovskii, 1972) . Thus thymines in a single-stranded DNA are sensitive to KMn04 modification, but become resistant when in¬ volved in a duplex (Hayatsu & Ukita, 1967) . Haner & Dervan (1991) have demonstrated that thymines on a single strand become less reactive toward KMn04 when the single strand is incorporated into a triple helix by occupying the major groove of a DNA duplex.
DMS reacts predominantly at the N-7 position of guanines (Maxam & Gilbert, 1980) . In a triple helix, the N-7 position of guanines in the Watson-Crick duplex is involved in hydrogen bonding to the third strand in both C+-GC and GGC base triplets; in the case of C+-GC triplets, guanines have been shown to become resistant to DMS modifica- tion (Johnston, 1988; Hanvey et al., 1988).
DEPC, which carbethoxylates purines (A > G) primarily at the N-7 position, is useful for probing adenine residues involved in triplex formation. In T-AT base triplets, adenines in the Watson-Crick strand show protection from DEPC modification because of the Hoogsteen hydrogen bonding involving the N-7 position (Johnston, 1988; Htun & Dahlberg, 1988; Hanvey et al., 1988). Similar protection is expected for the Watson-Crick adenine of the A-AT base triplet.
Oligonucleotide I (Figure 8) has the general form (Pu)nN4(Py)nN4(Py)n and is expected to form an intramolecular triple helix with PyPuPy base trip¬ lets. On the other hand, oligonucleotide II (Figure 8) has the general form of (Py)nN4(Pu)nN4(Pu)n and thus has a potential of forming an intramolecular triplex with Pu-PuPy base triplets.
Oligonucleotides III-V (Figure 8) consist of two different triplex-forming motifs fused together, i.e., these oligonucleotides can be considered as hybrids of oligonucleotides I and II. They belong to the general form (Pu)n(Py)nN4(Pu)ιt (Py)nN4(Py)n(Pu)n and can potentially form a triplex consisting of a block of PyPuPy base triplets connected to a block of Pu.PuPy base triplets. For clarity, each oligo¬ nucleotide is described in three segments: 5', middle, and 3' segments.
The results of KMn04 modification on oligonucle¬ otide I performed in 10 mM Tris-HCl (pH 7.0) and 0.1 mM EDTA (TE) buffer are shown in Figure 2 (Example IA) . Thymines in the middle segment (T12-T17) are less reactive toward KMn04 than thymines on either side (Figure 2, lane 2), consistent with the middle segment being base-paired to the 5' segment, making a WatsonCrick duplex. With increasing concentration of Mg++ ions, thymines in the 3' segment ^-T^; bracket) become more and more resistant to KMn04 modification and the protection of thymines in the middle segment becomes more pronounced (Figure 2, lanes 3-5). In 20 mM MgCl2 (Figure 2, lane 5), the overall reactivity pattern is consistent with an intramolecular triplex having T\AT base triplets, as expected, with only the thymines that occupy the putative loop regions (T9-Tu and T18-T22) being sensi¬ tive to KMn04 modification and thus providing an internal control for the modification. The first two thymines at the 5' end remain hyperreactive toward KMn04 throughout all conditions, indicating that these bases are unpaired possibly because of "fraying," i.e., breathing or transient un-pairing of normally paired bases at an end of a molecule.
In contrast to oligonucleotide I, oligonucleo¬ tide II has the potential to form an intramolecular triplex with A-AT base triplets. The reactivity patterns of oligonucleotide II in 50 mM sodium cacodylate (pH 7.1) buffer (Figure 3, Example IB) support a triplex conformation. In addition to the thymines occupying the two potential loops (T9-T12 and TJJ-T^) , the first four thymines (Tj-T4) at the 5' end are also reactive toward KMn04 (Figure 3, lanes 2-4, arrows), suggesting that these bases are at least transiently unpaired or frayed. The lesser reactivity of internal thymines in the 5 ' segment (T5-T8) suggests the formation of a Watson-Crick duplex within this region. The reactivity of oligo¬ nucleotide II toward DEPC in the same buffer is shown in lanes 6-8 (Figure 3) . All adenines of the 3'-segment are equally reactive, whereas those on the middle segment are not; A13-A16 are protected from DEPC modification, and A^A^ are reactive. The DEPC protection of A13-A16 residues can be attributed to the formation of A-AT base triplets. The reactivity pattern of these adenines does not change with the addition of Mg++ions, indicating that the formation of A-AT triplets does not require Mg++ ions. This finding is analogous to the behavior of G-GC base triplets, detection of which does not require Mg++ ions in the medium (Chen, 1991) . The reactivity of 17. 20 indicates that these adenines are not stably involved in the formation of A-AT triplets, apparent- ly because of fraying of the homologous Watson-Crick duplex in this region as deduced from KMn04 reactivi¬ ty.
Attempts to construct intermolecular, as opposed to intramolecular, duplex or triplex pairing schemes for either oligonucleotides I or II that were consistent with the observed reactivities were unsuccessful. For example, an inter olecular duplex structure for oligonucleotide I should result in protection of thymines next to the adenine tract (T9-T16) , not hyperreactivity as is observed.
Oligonucleotide III has two adjacent potential triple-helix forming motifs, one making TΑT and the other making A-AT triplets. Thymines T4-T7 on the 5' segment are unreactive toward KMn04 in 50 mM sodium cacodylate (pH 7.1) buffer (Example 1C; Figure 4A, lane 3) , suggesting that they are in a Watson-Crick duplex. In contrast to T4-T7, T^-Tjg on the 3 r segment are modified by KMn04 (Figure 4A, lane 3) , indicative of single-stranded character. With the addition of Mg++ ions to the medium, the reactivity of these reactive thymines (T^^g) is reduced (Figure 4A, lanes 4-6, bracket) , suggesting triplex formation with T-AT base triplets. In addition to T 2*~τ 26' thymine T21 shows Mg++-dependent resistance toward modification. Because it is flanked by two guanines in the loop, the protection is probably due to stacking with adjacent guanines induced by Mg++-dependentfolding. Such protection from KMn04 of thymines primarily residing in loops has been observed previously (Haner & Dervan, 1990) .
Oligonucleotide III was modified with DEPC in the same buffer to detect whether AΑT triplets are formed, in addition to TAT triplets; the results are shown in lanes 9-12 of Figure 4A (Example 1C) . Adenines 13-15 are protected from DEPC (open arrow¬ heads) , suggesting the formation of AAT base trip- lets. As observed for oligonucleotide II, the reactivity of these less-reactive adenines (A13-A1S) is not sensitive to the presence of Mg++ ions. Because it is located in the loop region, the enhanced reactivity of Au (bottom closed arrowhead) is expected, and it serves as an internal control for the modification. However, the high reactivity of A12 (a base at the extreme end of the helix) is unexpected and could be due to the distortion caused by the loop making it unavailable for base pairing. Adenines at the 2 ' end ( ^-hj.) are reactive whereas adenines at the 5' end (A^) are less reactive, as clearly seen in Figure 4B (bottom of the gel, lanes 1 and 2) , where DEPC modification was performed at different temperatures. In the absence of Mg++ ions the less reactive adenines (A13-A15; bracket) become reactive at approximately 37°C (Figure 4B, lane 3), suggesting the melting of AAT base triplets. The addition of Mg++ions raises the melting temperature, so that maximal reactivity is seen at 55°C instead of 37°C (Figure 4B, lane 9). Thus, the results indicate that Mg++ ions are not essential for the formation of A-AT base triplets, but their stability is enhanced by Mg++ ions. Taken together, the results of KMn04 and DEPC modifications suggest the presence of both PyPuPy (TAT) and PuPuPy (AAT) base triplets within oligonucleotide III in the presence of Mg++ ions.
Like oligonucleotide III, oligonucleotide IV also has the potential to form a triplex having juxtaposed Pu-PuPy and PyPuPy motifs, but with different base triplets (TAT and GGC) . The results of KMn04 modification of oligonucleotide IV are shown in Figure 5A (Example ID) . Thymines on the 3' segment (T^-T^) are reactive toward KMh04 in TE buffer (Figure 5A, lane 3, bracket) , indicating that they are in a single-stranded form. However, with the addition of Mg++ions, T^-T^ become less reactive (Figure 5A, lanes 4-6) , suggesting the formation of T-AT base triplets. On the other hand, at all Mg++ ion concentrations, T17-T20 remain less reactive, consistent with their involvement in a duplex. The reactivities of T9-T12 and T^-T^ do not change under any condition, indicating their singlestranded nature in the folded (Figure 5A, lane 6) as well as unfolded structure (Figure 5A, lane 3) .
The results of DMS modification of oligonucleo- tide IV performed in TE buffer at different tempera¬ tures are shown in Figure 5B (Example ID) . In the absence of Mg++ions, G13-G16 become more reactive at temperatures above 23"C (Figure 5B, lanes 1-6), suggesting the presence of G-GC base triplets at 23*C or below. In the presence of Mg++ ions, these guanines are less reactive at 23°C than they are in the absence of Mg++, indicating that, analogous to AAT base triplets, the stability of G-GC base trip¬ lets is also enhanced in the presence of Mg++ even though Mg++is not essential for their formation.
Thus the results of KMn04 and DMS modifications indicate the formation of a triple-helix containing both T-AT and G-GC base triplets in oligonucleotide IV. Comparison of the melting temperatures for oli- gonucleotides III and IV suggests that A-AT base triplets are more stable than G-GC base triplets.
The potential triple helix-forming regions of oligonucleotide V consist of mixed purines and mixed pyrimidines. Unlike the case for other oligonucleo- tides, the formation of an intramolecular triplex by oligonucleotide V is expected to be pH-dependent because of the involvement of C+-GC base triplets. The results of KMn04 modification of oligonucleotide V at different pH values are shown in Figure 6A (Example IE). At pH 4.5, both T26 and T28 on the 3' segment are protected from KMn04 modification (Figure 6A, lane 3, arrowheads) whereas at pH 6 and 7 these two thymines are modified. This pH-dependent reac¬ tivity is consistent with the formation of T-AT base triplets in the 3' segment at a pH permitting C+GC triplets to form. In 10 mM Mg++, T26 and T28 remain unreactive at both pH 6 and 7.
The formation of A-AT and GGC base triplets in oligonucleotide V is detected by reacting with DEPC and DMS, respectively (Example IE) . As seen in lanes 3-7 of Figure 6B, A14 and A16 are less reactive toward DEPC (open arrowheads) than A^ (closed arrowhead) , consistent with A14 and A16 being in¬ volved in A-AT base triplets and A^ residing in a loop. Both Aj and A4 are less reactive, consistent with their involvement in TAT base triplets. With DMS modification (Figure 6B, lanes 8-12) , G13 and G15 remain less reactive than the internal control G9 (in a loop; closed arrowhead) , suggesting the involve¬ ment of G13 and G15 in G-GC base triplets. Here again, the formation of A-AT and GGC base triplets is not dependent on Mg++ ions. Furthermore, as expected, their formation is independent of pH as shown by the identical reactivity patterns observed for both DEPC and DMS modifications at pH 4.5 and 7.0 (Figure 6B, compare lanes 4 and 5 with 6 and 7, and lanes 9 and 10 with 11 and 12) .
Figure 6C (Example IE) shows the melting of oligonucleotide V as monitored by DEPC reactivity. As was the case with Pu*PuPy base triplets in oligo¬ nucleotides III and IV, A«AT triplets (at A14 and A16) melt at a higher temperature in the presence of Mg++ ions (>70°C at 10 mM Mg++compared to <55°C without Mg++; Figure 6C, compare lanes 3-6 with lanes 7-10 (arrow heads)) . On the other hand, G3 and A4 show a transition to increased reactivity well below 70'C even in the presence of Mg++ ions, indicating the melting of C+-GC and T-AT base triplets. Therefore, in the presence of Mg++ ions, A-AT base triplets in this mixed purine tract appear to be more stable than T-AT base triplets. The different melting temperatures observed for AΑT base triplets generat¬ ed within oligonucleotides V and III could be due to a sequence effect (oligonucleotide V contains 50% GC) . In summary, the results of chemical modifica¬ tions of oligonucleotide V show the formation of intramolecular triplexes utilizing alternate strands of duplex DNA containing all four bases. Experiments performed in support of the present invention show that using two types of base trip¬ lets, Pu-PuPy and PyPuPy, a single strand consisting of oligopurine- and oligopyrimidine-blocks bind simultaneously to a duplex DNA, where the duplex contains adjacent tracts of oligopurines and oligo- pyrimidines. This binding is via both Pu-PuPy and PyPuPy base triplets, as exemplified in Figure la(iii) .
In Figure la(iii) , the oligopyrimidine block of the single strand is parallel to one purine tract of the duplex DNA and its oligopurine block is anti- parallel to the other purine tract on the alternate strand of the duplex. Unlike previous methods, where polarity considerations required the design of an oligonucleotide with an unnatural 3'-3' linkage (Home & Dervan, 1990) , the present invention requires no special junction for this polarity of binding between the oligopurine and oligopyrimidine blocks of the single strand occupying the major groove of both oligopurine and oligopyrimidine tracts. The above experiments support that triple-helix formation can occur as described above, at tandem oligopurine and oligopyrimidine tracts. The reac¬ tivity patterns of the above-discussed oligonucleo- tides, as well as the deduced structures for each, are summarized in Figure 7.
Two different hydrogen bonding schemes have been proposed for the A-AT base triplet (Beal & Dervan, 1991) , one with two hydrogen bonds involv- ing N-7 and N-6 of the Watson-Crick adenine, and the other with only one hydrogen bond forming at the N-6 of the same adenine. Because the Watson-Crick adenine is protected from DEPC, which reacts primar¬ ily at the N-7 of purines, the experiments performed in support of the present invention suggest that the A-AT base triplet has two hydrogen bonds involving both N-7 and N-6 of the Watson-Crick adenine.
Oligonucleotides III-V, containing tandem tracts of oligopurines and oligopyrimidines in different base combinations, exhibit chemical reactivity patterns that are in agreement with an intramolecular triplex structure forming both PuPuPy and PyPuPy base triplets simultaneously. In each case, the reactivity patterns described above are consistent with the bases of a single-stranded region occupying the major groove of the duplex hairpin, base-pairing to the purine tract in each Watson-Crick strand, as depicted in Figure 1 (A and B). The results of chemical modification performed at different temperatures suggest that Mg++ ions enhance the stability of PuPuPy base triplets, although their formation does not require Mg++.
The above experiments suggest that both PyPuPy and PuPuPy base triplets can be incorporated into the triplex by using a third strand consisting of oligopurine and oligopyrimidine blocks. This approach allows triplex formation at sequences consisting of tandem tracts of oligopurines and oligopyrimidines. Designing oligonucleotides to bind purine tracts of alternate strands appears to be a viable strategy for triplex formation involving any sequence of DNA, relaxing the restriction that target sequences be long (>10 nucleotides) oligo¬ purine tracts.
II. Diagnostic and Therapeutic Application of the Oligonucleotides of the Present Invention. A. Band-Shift Assays.
The third strand oligonucleotides of the present invention can be used in a number of diag- nostic and therapeutic applications. One diagnostic application of the oligonucleotides of the present invention is to provide a means of site-directed cleavage at a target duplex by coupling oligonucleo¬ tides to cleaving agents. One therapeutic applica- tion of the present invention is targeting a select¬ ed gene for inactivation, e.g., by suppression of mRNA synthesis from the target gene (Cooney et al., 1989; Orson et al., 1991; Postel et al. , 1991). In both of the above applications a defined oligonucle- otide is directed at the duplex DNA target to form a triple helix.
The triple-helix approach for targeting DNA has had limited use to date because of the requirement for long homopurine target sequences. Prior to the present invention, triplex formation at an oligo¬ purine*oligopyrimidine typically required a single strand consisting of only either pyrimidines or only purines; the ability to use alternating tracts of oligopurine and oligopyrimidine sequences, as described above, obviates this requirement.
Oligonucleotides of the present invention are tested for triplex formation with duplex target sequences using band shift assays (Example 2) . Typically, high percentage polyacrylamide gels are used for band-shift analysis where denaturing conditions (Ausubel et al. ; Sauer et al.; Sambrook et al.) are adjusted to reduce any background of non-specific binding.
In general a target duplex oligonucleotide is used initially for band shift analysis. The duplex target is radiolabeled and mixed with a third strand oligonucleotide which is being tested for its ability to form triplex structures with the target duplex. Oligonucleotides are evaluated for their ability to shift the mobility of the duplex oligonu¬ cleotide. Triplex formation is indicated in the band shift assay by a decreased mobility in the gel of the labeled triplex structure relative to the labeled duplex structure, i.e., triplexes appear as bands having apparent higher molecular weight. A number of controls are performed to assure the oligonucleotide specificity of triplex-forma¬ tion. First, pre-mixing the third strand oligonu¬ cleotide with a complementary DNA strand, prior to triplex formation, should block the ability of oli- gonucleotide to form a triplex with the radiolabeled duplex. The complementary strand added to the duplex alone should neither have an effect on the mobility of the radiolabeled duplex DNA, nor be able to block as complementary oligonucleotides when added to reaction mixtures after triplex formation has occurred. Second, oligonucleotides having similar purine and pyrimidine compositions to the third strand oligonucleotide, but which have a random mix of the purine and pyrimidines instead of to the sequence specified by the duplex target are prepared. The random sequence oligonucleotides should neither affect the mobility of labeled duplex target nor interfere with the band-shift caused by sequence-specific third-strand oligonucleotide.
Similar experiments can be employed in which the single strand oligonucleotides are radiolabeled and the duplex added to them. The same logic is used in establishing control experiments as outlined for the radiolabeled duplex DNA.
B. Cleaving Agents Covalently Attached to Third Strand Oligonucleotides.
Typically, inhibition of gene expression via triplex formation has been attempted only with olig¬ onucleotides carrying no chemical cleaving groups (Cooney et al. , 1989; Orson et al., 1991; Postel et al. , 1991). This is also true-for antisense inhibi¬ tion of gene expression (Matsukura et al. , 1989; Agrawal et al., 1989; Zamicnik et al. , 1986; Rittner and Sczakiel, 1991) . To be effective, antisense agents of this type must continuously bind to all the target molecules in such a way as to inactivate them. However, the oligonucleotides of the present invention can be equipped with chemical cleaving groups.
The advantage of using a chemical cleaving moiety is that, in addition to binding, the reagent permanently inactivates the target nucleic acid sequences. This method is applicable to any double strand DNA target, and can be also be applied to selected target RNA molecules (see below) . The present invention is particularly useful in target¬ ing viral genomes (free replicating or integrated) for inactivation.
An example of a family of such viruses is the family of human immunodeficiency viruses (HIV) , in particular, HIV-1, which appears to be the etiologi- cal agent responsible for AIDS.
Potential third strand oligonucleotides are tested for triplex formation as described above. The sequences of the oligonucleotides are chosen based on target sequences having tracts of oligo- purines-oligopyrimidines. For example, Figure 9A-9C illustrate three potential sites for targeting within the LTR region of HIV-1 with single-stranded oligonucleotides. Figure 10 shows the sequences of oligonucleotides A 1 (SEQ ID NO:9) and 2 (SEQ ID NO:10), B 1 (SEQ ID NO:ll) and 2 (SEQ ID NO:12), and C 1 (SEQ ID NO:13) and 2 (SEQ ID NO:14) designed to target sequences of Figure 9A, 9B, and 9C, respec¬ tively. Next to each of these oligonucleotides (Figure 10) is the general pattern of base triplets expected to form when triplexes are formed. Oligo¬ nucleotides K (SEQ ID NO:15), L (SEQ ID NO:16), and M (SEQ ID NO:17) are the control oligonucleotides (Figure 10) .
All three target sites are located in the control region of the LTR, i.e., upstream of the transcription initiation site and therefore do not interact with mRNA sequences to function as anti¬ sense mediators. The potential target sites A, B, and C (Figure 9) have different triplex forming motifs: Site A, consisting exclusively of purines is targeted with oligonucleotides A-l and A-2 (Figure 10) , which are capable of forming triplexes with Pu*PuPy and Py»PuPy base triplets, respective¬ ly. Site B consists of a tract of pyrimidine residues flanked by two purine tracts and is target¬ ed with oligonucleotides B-l and B-2 (Figure 10) , which have the correct polarities for triplex forma¬ tion. Site C has some pyrimidines buried in a highly purine-rich sequence, and oligonucleotides C-1 and C-2 (Figure 10) is directed toward site C. Oligonu¬ cleotides K, L, and M, each having incorrect polari- ty for triplex formation at sites A, B, and C, respectively, are used as controls. Test oligomers with and without phenanthroline are used to assess the effect of cleavage.
A number of cleaving agents may be used in the present invention including the following chemical moieties which are capable of cleaving nucleic acid substrates: phenanthroline (Chen et al., 1986, 1987; Francois et al. , 1989; Ebright et al. , 1990), Fe(II)-EDTA (Dreyer et al. , 1985; Dervan, 1986; Moser et al. , 1987; Maher et al. , 1989; Sluka et al., 1987), Cu(II)-bipyridine, Cu(II)-terpyridine, and Zn(II) (Modak et al. , 1991; Eichhom et al., 1971; ; Breslow et al. , 1989). These chemical cleaving moieties can be employed in the present invention as exemplified below with reference to the phenanthroline moiety.
The cleaving agent 1,10-phenanthroline is attached to the third strand oligonucleotides as described in Example 3.
The ability of test oligonucleotides to form triplexes and carry out cleavage at their designated target sites is assayed by determining the site-specific cleavage induced at the target se¬ quences by test oligonucleotides equipped with the phenanthroline moiety (Example 4) . For the oligonu- cleotides shown in Figure 10, pHIV-lCAT is linear¬ ized with, end-labeled, and subjected to a second restriction digest to obtain a uniquely labeled DNA fragment. After gel purification, this DNA fragment is mixed with a phenanthroline modified oligonucleo- tide. Cleavage products are resolved on sequencing gels along with the products of sequencing reac¬ tions. This method maps the site of triplex forma¬ tion, and provides an indication of the cleavage efficiency (detected by counting the radioactivity of excised gel bands) ; the method allows the quantitation of the efficiency of triplex formation. In vitro triplex formation can also be tested using the band shift assay described above, where cleavage of the duplex target is evidenced by increased mobility of the fragments of the labeled duplexes in the gel. For these assays polyacryl- amide gels run under DNA-denaturing conditions (Ausubel et al.; Sambrook et al.) can be used.
To examine the ability of the phenanthroline- coupled-oligonucleotides to inhibit gene expression by in vivo triplex formation, transient expression of the CAT gene under the direction of HIV-1 LTR in HeLa cells is used. HeLa cells are transfected with pHIV-1 CAT, using the DEAE-dextran technique (Queen and Baltimore, 1983) or Ca3(P04)2 technique. Oligo- nucleotides carrying phenanthroline are complexed with CuS04 before they are introduced to the cell medium. Approximately twelve hours after transfec- tion, the cells are incubated with a phenathroline- coupled-oligonucleotide, as described by Postel et al. (1991) . Mercaptopropionic acid or ascorbic acid are then supplied to the medium approximately 12 hr after the oligonucleotide treatment.
Mitomycin C is added to the medium to induce CAT expression; since the HIV-1 LTR is under the influence of NF-kB, the expression of CAT activity can be induced by treating with either ultraviolet light or mitomycin C. Cells are harvested at 12 and 24 hr after mitomycin C addition, and CAT activities determined as described by Gorman et al. (1982) . CAT expression is compared to controls including cells that have been exposed to control phenanthro- line-coupled-oligonucleotides (K, L, and M) , phenan- throline-coupled-oligonucleotides (SEQ ID N0:9-SEQ ID NO:17) without complexing to CuS04, and cells without oligonucleotide treatment. Successful cleavage of the target by the duplex-targeted third strand oligonucleotide reduces levels of CAT expres- sion relative to controls, in particular, relative to phenanthroline-coupled-oligonucleotides (K, L, and M) complexed to CuS04.
Other HIV-reporter gene constructs can be used in the above experiments (e.g., Cross et al.), including HIV-LTR constructs containing a luciferase (de Wet et al.) reporter gene.
C. Oligonucleotide Inhibition. In addition to the above-described site direct¬ ed cleavage of target DNA duplexes by the oligonu¬ cleotides of the present invention, oligonucleotides can be constructed to target duplex DNA regions in order to block expression — without cleavage. Target sites can be chosen in the control region of the genes, e.g., in the transcription initiation site or binding regions of regulatory proteins (Helene and Toulme, 1990; Birg et al., 1990; Postel et al. , 1991; Cooney et al. , 1988). These oligonucleotides can also interact with mRNA sequences to function as antisense mediators (see below) .
Single-stranded DNA can also be used as a target nucleic acid for oligonucleotides of the present invention. For example, two oligonucleo¬ tides can be used for target-directed binding. Alternately, oligonucleotides similar to those described below (TDAs) can be used for the binding or binding/cleavage of target sequences contained in single-stranded DNA molecules.
D. Triplex-Directed Antisense Oligonucle¬ otides.
Single-stranded oligonucleotides of the present invention can also be used in an anti-sense expres¬ sion inhibition technique, where the single-stranded oligonucleotides are capable of forming triplex structures with target m NAs (Giovannangeli et al., 1991) . This triplex-directed antisense (TDA) approach may be more effective than a duplex (con¬ ventional antisense approach) in arresting biologi¬ cal processes such as translation and reverse transcription. Triplex formation in this fashion is highly selective and of high affinity and may not be a substrate for enzymes such as helicases. The action of such helicases can be a potential problem in the conventional antisense approach. Experiments performed in support of the present invention, see intramolecular triplexes described above, indicate the feasibility of triple helix formation between a single-stranded RNA and a singlestranded DNA which contains complementary regions capable of forming a duplex. The complexa- tion of single-stranded oligonucleotides with circular oligonucleotides has been studied (Kool, 1991) and complexation by circular oligonucleotides that can form triplexes with the single strands has been found to be more selective and stable than by the linear complementary oligonucleotides.
The TDA approach of the present invention is applicable to any target mRNA molecule; an example of targeting HIV mRNA is presented below. Three TDA target sites (D, E, and F) within the 5786-8887 nucleotide region of the HIV-l genome are presented in Figure 12A. The figure also shows a target region for each mRNA sequence (underlined) . TDA oligonucleotides directed to these sequences are presented as oligonucleotides Dl, D2, El, E2, and Fl, F2 in Figure 12B. Oligonucleotides complementa¬ ry to the target regions, i.e., oligonucleotides designed to function as conventional antisense agents, are presented as oligonucleotides Dx, Ex, and Fx in Figure 12B.
For each of these target mRNA sequences (Figure 12A, underlined) , oligonucleotides capable of forming triplexes are synthesized. Two different triplexes, one with Pu»PuPy and the other with Py»PuPy base triplets, are possible for each target (Figure 12B) . To avoid nonspecific interactions with loop residues of hairpins, connecting sequences ("loops") are synthesized with the "UNI-LINK AMINO- MODIFIER" (Clonetech, Palo Alto CA) , which lacks a base and a sugar attached to the phosphate. Because of the continuity and the proper interphosphate spacing of the backbone rendered by "UNI-LINK AMINOMODIFIER" triplex formation is not be affect¬ ed. In Figure 12B, "X" indicates an abasic "UNI-LINK AMINOMODIFIER" site.
The in vivo effect of TDA oligonucleotides are assayed by detecting the expression of gpl20 (enve¬ lope glycoprotein) of HIV. HeLa T4+ cells (AIDS Research and Reference Program) are transfected with the pDOLHIVenv (available from the AIDS Research and Reference Program) plasmid and selected for β-kanamycin resistance. The pDOLHIVenv plasmid has open reading frames for Env, Tat, and Rev proteins (nucleotides 5786-8887 of the HIV-1 genome) . jβ-Kanamycin-resistant cells are picked and expanded. These cells are then incubated with a selected TDA oligonucleotide for 24 hr. The level of expression of envelope proteins is assayed by a syncytium (giant cell) formation assay carried out in microtiter plates (Putney, et al. , 1989). Because syncytium induction by HIV is mediated by the interaction of the CD4 molecule with gpl20 on the surface of cells, the number of syncytia is directly proportional to the amount of expressed gpl20.
To compare the effectiveness of the TDA oligo¬ nucleotides and conventional antisense oligonucleo- tides, oligonucleotides D2, E2, and F2, which are capable of direct binding to mRNA, are used as controls in gpl20 expression experiments. Alternatively, an indirect immunofluorescence assay is employed to detect the level of gpl20 expression. In this case, an antiserum to gpl20RF (available from the AIDS Research and Reference Program) is used as the primary antibody in a standard indirect immunofluorescence antigen detec¬ tion assay (Harlow et al.). Rhodamine isothiocya- nate-conjugated rabbit anti-goat immunoglobulin G (Pierce, Rockford, IL) is used as the secondary detection antibody.
TDA oligonucleotides can be targeted to regula¬ tory regions, as described above, or to other regions, such as splice sites in pre-mRNA molecules.
E. Relative Propensities of Formation of Different Base Triplets.
In addition to A»AT and G»GC, several unusual base triplets have been documented. These include
A+«GC, base triplets in DNA and I»AU and I*GC in RNA. Triple helix formation is extremely sequence- specific and bases that can form hydrogen bonds with Watson-Crick base pairs can form triplexes. Howev¬ er, the relative stabilities which may vary from one base triplex to the other is important in designing single strand probes for triplex formation. The affinity cleaving assay described above is used to detect the relative propensity of triplex formation involving oligonucleotides containing the above unusual base triplets. For example, the propensity of triplex forma¬ tion by I*GC with respect to that of G»GC is assayed by comparing the cleavage efficiency induced by oli¬ gonucleotide II and Gl (Figure 15B) . In these two cases the polarity of the third strand as well as the type of neighboring base triplets would be the same except for variants, i.e., I»GC and G»GC base triplets. Thus the difference of cleavage efficien¬ cy induced by these two oligonucleotides simply reflect the propensity of triplex formation by I»GC and G»GC base triplets. Similarly, the propensity of the formation of I»AT triplet with respect to that of A«AT is be assayed using oligonucleotide 12 and Al (Figure 15B) .
Because I*GC and I»AT triplets have been documented, oligonucleotide II will be used to detect targeting in the duplex sequences shown in
Figure 15A.
F. General Considerations Concerning the Oli¬ gonucleotides of the Present Invention.
The oligonucleotides of the present invention comprise tandem, alternating tracts of purine and pyrimidine sequences, where the length of each tract may vary but contains at least one purine or pyrimi¬ dine base (Example 4B) . Typically, the tracts of purines and pyrimidines contain at least 4 purine bases followed by at least 4 pyrimidine bases. The number of bases in the tracts of purines and pyrimi¬ dines do not have to be equal, for example, a tract of 4 purines may be attached to a tract of 9 pyrimi¬ dines, or the oligonucleotide may comprise tandem tracts of 4 purines, 9 pyrimidines, 2 purines, 5 pyrimidines. The typical length of targets for the oligonucleotides of the present invention are from about 8 nucleotides to about 60 nucleotides; the length of the target is used, in part, to determine target specificity and triplex stability. Cellular uptake of the 32P-labeled oligonucleo¬ tides is evaluated essentially as described by Postel et al. Stability of the oligonucleo¬ tides in vivo can be evaluated by freeze-drying aliquots from the medium and from the nuclear and cytoplasmic fractions and analyzing the resuspended aliquots on 12% sequencing gels (Postel et al.).
The delivery of any of the oligonucleotides of the present invention into cells can be facilitated using established techniques such as scrape loading, electroporation, and microinjection. Alternatively, oligonucleotides can be encapsulated within lipo¬ somes and these loaded liposomes incubated with cells.
The ends of oligonucleotides can be protected by attaching intercalators; this has been shown to make oligonucleotides resistant to exonucleases and facilitates cell permeation (Cazenave et al., 1987) . The linking of intercalators improves the exonucle- ase resistance of oligonucleotides and further provides additional binding energy (because of intercalation) without perturbing the specificity of recognition of complementary sequences. Backbone modifications such as methylphosphonates and phos- phothioates not only make oligonucleotides endonu- clease resistant, but the neutral backbones also facilitate cell permeation (Matteucci et al. (1991) ; Miller et al.) . Oligonucleotides at the 5' and 3' ends can be derivatized with two different linking molecules using different chemistry. Such derivatization is performed by using "3'-AMINE-ON CPG" (Clontech) solid supports to incorporate a functional amino group at the 3' end, and incorporating a thiol group at the 5' end as described above. Because deprotec- tion procedures for the two functional groups are different and independent from each other, derivati¬ zation at the two ends is performed at two stages of oligonucleotide synthesis.
TDA oligonucleotides can be derivatized at one or both ends to modify the oligonucleotides to provide a cleavage function, as described above; RNA targets can be cleaved using the cleaving moieties described above, including 1,10 phenanthro¬ line. For cleavage reactions carried out in cells in culture, the intracellular reduction potential can be modulated using N-acetyl cysteine, which increas¬ es the intracellular glutathione level (Roederer et al. , 1990; Kalebic et al. , 1991). Such manipulation of the intracellular reduction should assist in keeping, for example, a copper atom of a cleaving agent in the reduced state.
The level of expression of cytoplasmic mRNA corresponding to target sequences can be evaluated by standard methods including: Northern analysis using a target mRNA specific probe (Ausubel et al. ; Sambrook et al.; Postel et al.) ; dot-blot hybridiza¬ tions using target mRNA specific probes (Ausubel et al.); and polymerase chain reaction (Mullis; Mullis et al.) using target mRNA specific probes.
For increasing binding affinity of oligonucleo¬ tides for their targets, substitution of Br5U (5- bromo-uracil) for thymines in the third strand increases binding affinity when a triplex is formed. Therefore, thymines that form Hoogstein hydrogen bonds can be replaced with Br5U during chemical synthesis. Further, the substitution of Me5C (5- methyl-cytosine) in place of cytosines in the third strand is useful for effective targeting at physio¬ logical pH.
III. Utility.
The ability of the oligonucleotides of the present invention to recognize targets having combinations of tracts of purines and pyrimidines provides flexibility for techniques based on triplex formation, including third strand inactivation of a target duplex gene and anti-sense inactivation (Helene and Toul e, 1990) .
Further, the cleaving reagents of the present invention provide means for a method of cleaving RNA targets at specific sites. Such cleavage is useful for the analysis of RNA structure and function as well as diagnostic analyses. One example of a diagnostic application is to isolate RNA from a cell infected with a particular RNA virus. Total or poly-A+ RNA (Ausubel et al.) is end labeled. The RNA is then isolated away from free label and the amount of incorporated label estimated, for example, by scintillation counting. The labeled RNA is then treated with an RNA cleaving agent, such as a TDA oligonucleotide combined with a chemical cleaving moiety, and the amount of liberated label is used as an indicator of the concentration of RNA contain the RNA-binding protein cognate binding site. The cleaving reagents of the present invention are particularly desirable for use with DNA virus targets and RNA virus targets or their pro-viral DNA forms: for example, cleaving HIV genomic RNA or pro-viral DNA. Oligonucleotides of the present invention, capable of forming triple-helix structures with single-strand or duplex DNA, can also be labeled and used as probes, or, when carrying cleaving agents, can be used as site-specific cleaving agents. The cleaving reagents of the present invention are also useful in a method of inhibiting expression of RNA viral (e.g., HIV) antigens in cells infected with the virus. In this application, infected cells are exposed to an oligonucleotide (DNA duplex directed, antisense, or TDA) modified to contain a cleaving moiety (i.e., the reagent), at a reagent concentration effective to produce reduction in viral antigen expression in the infected cells (Wang et al., 1988, 1989; Crowe et al., 1990). Examples of such reagents for anti-HIV agents have been described above. A combined use of RNA cleaving (or inhibiting) oligonucleotides combined with the above-described DNA duplex directed cleaving (or inhibiting) oligo¬ nucleotides may provide a two-pronged therapeutic attack against viral diseases by providing cleavage of viral RNA and DNA or pro-viral DNA genomes. A major advantage of targeting the DNA pro-virus associated with an RNA virus is that typically only one, or a few copies, of integrated, transcriptio- nally active DNA are present per cell in contrast to many copies of mRNA which may be present in an infected cell (Soma et al. , 1988) .
The following examples illustrate, but in no way are intended to limit the present invention.
Materials and Methods Oligonucleotides were synthesized on an Applied Biosystems Model 381 B automated DNA synthesizer (Applied Biosystems, Foster City CA) using cyano- ethyl phosphoramidites. After deprotection, they were purified by electrophoresis on denaturing 20% polyacrylamide gels (Ausubel et al. ; Sambrook et al.) . Purified oligonucleotides were labeled at 5' ends using T4 polynucleotide kinase (United States Biochemicals, Cleveland OH) and 32P-γ-ATP (Du Pont-NEN, Boston MA) according to the method of Maxam & Gilbert (1980) . Unincorporated ATP was separated (Ausubel et al. ; Sambrook et al.) from labeled oligonucleotides by passing the labeling reaction mixture through two successive "SEPHADEX G-50" spin columns (Pharmacia, Piscataway NJ) equilibrated in 10 mM Tris-HCl and 0.1 mM EDTA (pH 7.5).
Modification of oligonucleotides usingpotassi¬ um permanganate (KMn04) was performed essentially as described by Rubin & Schimid (1980) . A radiolabeled oligonucleotide (approximately 100 ng) was equili¬ brated in 9 ml. of the reaction buffer (see below) at 4'C for 10 min, then 1 ml. of 5 mM KMn04 (freshly diluted from a 100 mM stock stored at 4'C) was added. After 10 min of incubation at 4'C, the reaction was stopped by adding 2 ml. of neat allyl alcohol.
Modification of oligonucleotides using diethyl pyrocarbonate (DEPC) was performed essentially as described by Johnston & Rich (1985) or Herr (1985) . An end-labeled oligonucleotide (approximately 100 ng) was equilibrated in 97.5 ml. of a reaction buffer (see below) for 10 min at 4°C. Then 2.5 ml. of DEPC (Aldrich, Milwaukee WI) was added, vortexed briefly, and the incubation was continued for 30 min.
Modification of oligonucleotides using dimethyl sulfate (DMS) was performed essentially as described by Maxa & Gilbert (1980) . One microliter of a 1:200 dilution of DMS in water (freshly prepared) was added to an end-labeled oligonucleotide (approx¬ imately 100 ng) equilibrated in 9 ml. of reaction buffer (see below) at 4'C, and the modification was carried out for 10 min at the same temperature.
For modifications performed at higher tempera¬ tures than noted above, the reaction times were decreased accordingly in order to control excess modification. The modification reactions were stopped by two ethanol precipitations except in the case of KMn04, as noted above.
DNA pellets were thoroughly washed with 70% ethanol, dried, and treated with hot piperidine (1 M, 90*C for 30 min) . KMn04-modified DNA was treated with piperidine directly, without ethanol precipita¬ tion. After removing piperidine by vacuum evapora- tion, DNAs were resuspended in formamide loading buffer, and the cleavage products were resolved on 20% sequencing gels. Sequencing reactions on oligo¬ nucleotides were performed according to Williamson & Celander (1990) .
Example 1
Intramolecular Triplex Formation
A. KMn04 reactivity of oligonucleotide I. KMn04 modification was performed in 10 mM Tris-HCl (pH 7.0) and 0.1 mM EDTA (TE) buffer at 4'C in the absence (Figure 2, lane 2) and presence of MgCl2 (Figure 2, lanes 3-5, concentrations as indicated) . Figure 2, lane 1 is the A+G sequencing reaction of oligonucleotide I (Maxam and Gilbert) . Modification of thymines within the bracket, "{" indicated in lane 1, is sensitive to MgCl2. In Figure 2, the open and closed bars, indicated on the right, represent WatsonCrick duplex region; single lines show single-stranded regions; and the hatched bar indicates the third "strand" of the triplex.
B. KMn04 and DEPC reactivity of oligonucleotide II. Both modifications were performed at 4°C in 50 mM sodium cacodylate buffer (pH 7.1) with or without MgCl2. In Figure 3, lanes 1 and 5 are the A+G sequencing reaction for oligonucleotide II; lanes 2-4, KMn04 modification; and, lanes 6-8, DEPC modifi- cation. MgCl2 concentrations were as shown in the figure. Closed arrows show hyperreactive thymines (T2-T4) at the 5' end of the oligonucleotide. Open arrowheads show adenines (A13-A16) that are protected from DEPC modification, whereas closed arrowheads represent reactive adenines (A^-A^) . Helical and loop regions are designated as described above for Figure 2. C. KMn04 and DEPC reactivity of oligonucleotide
III.
1. KMn04 and DEPC Modifications,
KMn04 (Figure 4A, lanes 3-6) and DEPC (Figure 4A, lanes 9-12) modifications performed in 50 mM sodium cacodylate buffer (pH 7.1) at 4°C. The bracket (]) indicates thymines whose reactivity pattern is sensitive to MgCl2. In Figure 4A, lanes 1 and 7 are the G sequencing reaction, and lanes 2 and 8 are the A+G sequencing reaction. MgCl2 concen¬ trations were as indicated in the figure. Closed arrowheads indicate adenines that are reactive toward DEPC, whereas those indicated by open arrow¬ heads are protected.
2. DEPC reactivity as a function of tempera¬ ture.
DEPC reactivity as a function of temperature was examined in the absence (Figure 4B, lanes 1-6) and presence (Figure 4C, lanes 7-10) of 10 mM MgCl2. Temperatures were as indicated at the top of the figure. Helical and loop regions are as described above for Figure 2.
D. KMn04 and DMS reactivity of oligonucleotide IV.
1. KMn04 modification.
KMn04 modification performed in TE buffer at 4'C in the absence (Figure 5A, lane 3) and presence (Figure 5A, lane 4) of 5 mM MgCl2. In Figure 5A, lane
1 is the G sequencing reaction; lane 2, is the A+G sequencing reaction.
2. DMS reactivity as a function of tempera¬ ture. DMS reactivity as a function of temperature was examined in the absence (Figure 5B, lanes 1-5) and presence (Figure 5B, lanes 6-10) of 10 mM MgCl2. Temperatures were as indicated at the top of the figure. Bracketed bases (]) are affected by MgCl2 concentration, indicated in Figure 5A, or tempera¬ ture, indicated in Figure 5B. Helical and loop regions are as described above for Figure 2.
E. Reactivity of oligonucleotide V toward KMn04, DEPC and DMS.
1. KMh04 reactivity.
KMn04 reactivity was examined in a buffer containing 25 mM NaOAc and 25 mM Tris-HCl at pH 4.5 (Figure 6A, lane 3), pH 6.0 (Figure 6A, lane 4) and 7.0 (Figure 6A, lane 5) at 4'C. 2. DEPC and DMS reactivity.
DEPC (Figure 6B, lanes 3-7) and DMS (Figure 6B, lanes 8-12) reactivity at 4°C in the same buffer; pH and MgCl2 were as indicated. Bases indicated by closed arrowheads are reactive toward the chemical probes, whereas those indicated by open arrowheads are protected.
3. DEPC reactivity as a function of tempera¬ ture. DEPC reactivity in pH 7.0 buffer was examined as a function of temperature in the absence (Figure 6C, lanes 3-6) and presence (Figure 6C, lanes 7-10) of 10 mM MgCl2. Temperatures were as indicated at the top of the figure. Arrowheads designate ade- nines whose reactivities are temperature-dependent. In each panel of Figure 6, lane 1 shows the G sequencing reaction and lane 2 shows the A+G se¬ quencing reaction. Helical and loop regions are as described above for Figure 2.
Example 2 Band Shift Assays Triplex formation is analyzed essentially as described by Roberts et al. (1991) . Figure 9A shows a sample target duplex sequence (SEQ ID NO:6). A representative target sequence to be used for triplex formation is shown in bold; Figure lOA-1 and 10A-2 illustrate the sequences of sample third strand oligonucleotides (SEQ ID NO:9 and SEQ ID NO:10) .
The two oligonucleotides labeled A-l and A-2 both have sequences amenable to triple strand formation. Both oligonucleotides bind in the major groove to the top strand presented in Figure 9A; oligonucleotide Al is anti-parallel in the major groove and oligonucleotide A2 is parallel in the major groove. Oligonucleotides are tested for triplex forma¬ tion in band shift assays on non-denaturing high percentage polyacrylamide gels. Briefly, the two strands of duplex oligonucleotide shown in Figure 9A are each synthesized, and then annealed to form the duplex DNA molecule shown in Figure 9A. Typically the annealing reaction involves mixing the two purified oligonucleotides in IX TE+50 buffer (10 mM Tris HCl pH 7.4, 1 mM EDTA, 50 mM NaCl) , or other suitable buffer (Sauer et al.) . The annealing mixture is heated to approximately 85°C and cooled slowly to room temperature. This duplex DNA is then end-labeled using polynucleotide kinase and 7~32P-ATP (Ausubel et al.) as described above. The radiolabeled duplex is then mixed individu¬ ally with each third strand oligonucleotide being tested in 20 mM HEPES, pH 7.1, 50 mM NaCl, 5mM spermine. In general, the oligonucleotide single- strand is added in vast excess (e.g., 10 micromolar) to the 32P-labeled duplex oligonucleotide (e.g., 1.0 nM) . Reaction volumes are typically 15-20 microli- ters. The samples are then loaded on a 20% acryla- mide non-denaturing gel, acrylamide/bisacrylamide 19:1, formed using 20 mM HEPES, pH 7.1 buffer. The running buffer of the gels is (same hepes 20 mM , 7.1. The gels are typically run at 6V/cm at 4°C.
The gels are dried and exposed to X-ray film. The formation of triplex complexes is detected by a band shift, by reduction in the intensity of the duplex band and the appearance of a higher molecular weight band, which contains the duplex complexed to the test oligonucleotide to form the triplex struc¬ ture.
For each oligonucleotide the concentration of duplex can be held constant and the concentration of the test oligonucleotide varied over a concentration range to give an idea of concentration effects associated with the test oligonucleotide. Any oli¬ gonucleotide which potentially can form triplex structures with a given target duplex, can be tested as described above.
Example 3 Modification of Third-Strand Oligonucleotides Using 1. 10 Phenanthroline During the chemical synthesis, each oligonucle- otide is synthesized with a thiol group at the 5' end using the "C6-THIOL MODIFIER" reagent from Clonetech (Palo Alto, CA) , according to the manufac¬ turers instructions. Briefly, the oligonucleotides are deprotected after synthesis with NH4OH and then treated with silver nitrate to expose the thiol group. The oligonucleotide is immediately reacted with 5-iodoacetamido 1,10-phenanthroline. Commer¬ cially available 5-nitro-l,10-phenanthroline (Sigma) is converted to 5-iodoacetamido-l,10-phenanthroline (Chen et al., 1986, herein incorporated by refer¬ ence) (Figure 11) to covalently link 1,10-phenan- throline to the oligonucleotide.
After the addition of the phenanthroline moiety, the resulting oligonucleotides are separated from un-reacted iodo compound by passing the reac¬ tion mixtures through "SEPHADEX G-50" spin columns (Pharmacia, Piscataway NJ) .
Phenanthroline attached to an oligonucleotide binds cupric ion and this complex can be used to cleave DNA. In the presence of a reducing agent the bound cupric ion is reduced to cuprous ion, which reduces molecular oxygen to produce hydrogen perox¬ ide. The E^Oj reacts with the cuprous complex to form a copperoxo species that is directly responsi- ble for cleavage (Sigman, 1990) .
An alternative but chemically analogous system (Dreyer et al., 1985; Dervan, 1986; Moser et al., 1987; Maher et al. , 1989) utilizes EDTA-chelated iron tethered to an oligonucleotide to cleave DNA.
The above cleaving agents were also described in co-owned, co-pending U.S. Application Serial No. 07/808,452, herein incorporated by reference.
Example 4 In Vitro Cleavage of Target Duplexes A. pHIV-lLTR-CAT Substrate. To evaluate the ability of the phenanthroline- coupled-oligonucleotide to mediate in vitro cleav¬ age, pHIV-lLTR-CAT (S. Miller, SRI International, Menlo Park CA) is linearized with Hindlll (Boehrin- ger Mannheim, Indianapolis IN) , end-labeled with 32P-γ-ATP (NEN) and polynucleotide kinase, and subjected to a second restriction digest to obtain a uniquely labeled DNA fragment, i.e., one carrying label only at one end of the linearized molecule. The pHIV-lLTR-CAT fragment carrying the target sequence and end-label is gel purified (Ausubel et al.; Sambrook et al.) .
After gel purification, this DNA fragment is mixed with phenanthroline modified oligonucleotide in a buffer containing 10 mM Tris-HCl, 100 mM NaCl, 500 mM spermine, and 20 mM MgCl2. The pH of the buffer is adjusted depending on the sequence of the target (a lower pH is used for the formation of C+*GC base triplets). After incubation at 20°C for 30 min, cleavage is initiated by adding CuS04 (to 10 mM) and mercaptopropionic acid (to 2.5 mM) . Cleavage products are resolved on sequencing gels along with the products of sequencing reactions for the pHIV- 1LTR-CAT target fragment.
B. Alternative Substrates The oligonucleotides shown in Figure 14A are used to evaluate the lengths of purine and pyrimi- dine tracts which are useful in the practice of the present invention.
The oligonucleotide sequences (Figure 14A) are inserted into pUC18 (Bethesda Research Laboratories, Gaithersburg MD) between the EcoRI and BamHI sites. The recombinant plasmids are purified and used as substrates for affinity cleaving assay. For the affinity cleaving assay, single-stranded oligonucle¬ otides covalently bound to Cu(II)-1,10-phenanthro- line are used as third strands. Two different third strands are possible for each target sequence; both candidates are synthesized and studied. For exam¬ ple, the two oligonucleotides shown in Figure 14B can form triplexes with the duplex target sequence #3 shown in Figure 14A.
The cleavage reactions are carried out and analyzed essentially as described above.
While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.
SEQUENCE LISTING (1) GENERAL INFORMATION:
(i) APPLICANT: Jayasena, Sumedha D. Johnston, Brian H.
(ii) TITLE OF INVENTION: Triple Helix Formation at (PuNPyN)-(PuNPγN) Tracts
(iii) NUMBER OF SEQUENCES: 53
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SRI International
(B) STREET: 333 Ravenswood Avenue
(C) CITY: Menlo Park (D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94025
(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:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/826,934
(B) FILING DATE: 21-JAN-1992
(vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 07/808,452
(B) FILING DATE: 13-DEC-1991
(viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Fabian, Gary R. (B) REGISTRATION NUMBER: 33,875
(C) REFERENCE/DOCKET NUMBER: P-3141 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 859-4550
(B) TELEFAX: (415 ) 859-3880 ( 2 ) INFORMATION FOR SEQ ID NO : l :
( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 32 base pairs ( B ) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Oligonucleotide I, FIGURE 8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
AAAAAAAATT TTTTTTTTT TTTTTTTTTT TT 3
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Oligonucleotide II, FIGURE 8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
ττττττττττ TTAAAAAAAA TTTTAAAAAA AA 3
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Oligonucleotide III, FIGURE 8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
AAATTTTGTT AAAAATTTGG TGGTTTAAAA 3
(2) INFORMATION FOR SEQ ID NO: :
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Oligonucleotide IV, FIGURE 8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
AAAACCCCTT TTGGGGTTTT CTTCTTTTGG GG 32
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: Oligonucleotide V, FIGURE 8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GAGATCTCGT TTGAGATCTC TTTACTCTAG AG
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SEQUENCE A, FIGURE 9
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TTTAAAAGAA AAGGGGGGAC TGG 2
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SEQUENCE B, FIGURE 9 (xi) SEQUENCE DESCRIPTION: SEQ. ID NO:7:
GCTGGGGACT TTCCAGGGAG GCGT
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: SEQUENCE C, FIGURE 9
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CCTGGGCGGG ACTGGGGAGT GGCGAGCCC 2
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, A-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
AGGGGGGAAA AGAAAA 1 ( 2 ) INFORMATION FOR SEQ ID NO: 10 :
( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 16 base pairs ( B ) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, A-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
TTTTCTTTTC CCCCCT
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, B-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GGAGGGACCT TTCAGGGG
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, B-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
CCCCTGAAAG GTCCCTCC 18
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, C-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GAGGGGAGAG GGGAGAGGGG GGG 23
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, C-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
CCCGCCCTGA CCCCTCACCG CTC
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, K
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
AAAAGAAAAG GGGGGA 1
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, L (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GGGGATCCCT TAGGGAGG
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 10, M
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
GGGTGGGATC GGGGAGCGGT GGAGAG
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12A, (D)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GGTAGAAGAG GAGGAAAAAA GGAAAAACTG 3 (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12, (E)
(xi)' SEQUENCE DESCRIPTION: SEQ ID NO:19:
CCCGAAGAAA TAGAAGAAGA AGGTGGAGAG CGAGGAGAGA CAGATCC 4
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12A, (F)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
CCACTTTTTA AAAGAAAAGG GGGGACTGGA AGGGCTA 3
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, D-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
TCTTCTCCTC CTTTTTTCCT TTTTNNNNNT TTTTCCTTTT TTCCTCCTCT TCT
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, D-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
AGAAGAGGAG GAAAAAAGGA AAAANNNNNT TTTTCCTTTT TTCCTCCTCT TCT 5
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 87 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, E-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CTTCTTTATC TTCTTCTTCC ACCTCTCGCT CCTCTCTGTC TNNNNNTCTG TCTCTCCTCG 6
CTCTCCACCT TCTTCTTCTA TTTCTTC 8
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, E-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GAAGAAAAAG AAGAAGAAGG AGGAGAGGGA GGAGAGAGAG ANNNNNTCTG TCTCTCCTCG 6
CTCTCCACCT TCTTCTTCTA TTTCTTC 8
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 67 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, F-l
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GAAAAATTTT CTTTTCCCCC CTCTCCTTCC CNNNNNCCCT TCCAGTCCCC CCTTTTCTTT
TTTTTTC
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 67 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, F-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GAAAAAAAAA GAAAAGGGGG GAGAGGAAGG GNNNNNCCCT TCCAGTCCCC CCTTTTCTTT 6
TAAAAAG 6
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, Dx
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
TCTTCTCCTC CTTTTTTCCT TTTT 2
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, Ex
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CTTCTTTATC TTCTTCTTCC ACCTCTCGCT CCTCTCTGTC T 4
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 12B, Fx (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CTTTTTTTTT CTTTTCCCCC CTGACCTTCC C 3
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
AATTCGGGGG GGGGGTTTTT TTTTTG 26
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
GCCCCCCCCC CAAAAAAAAA ACCTAG 26 ( 2 ) INFORMATION FOR SEQ ID NO: 32 :
( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 26 base pairs (B ) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
AATTCGGGGG GTTTTTTTTG GGGGGG 2
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
( i) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #2
( i) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GCCCCCCAAA AAAAACCCCC CCCTAG 2
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
AATTCGGGGG TTTTTGGGGG TTTTTG
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE; FIGURE 14A, TARGET SEQUENCE #3
( i) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GCCCCCAAAA ACCCCCAAAA ACCTAG 2
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
AATTCGGGGT TTTGGGGTTT TGGGGG
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
GCCCCAAAAC CCCAAAACCC CCCTAG
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairε (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #5 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
AATTCGGGTT TTGGGTTTGG GTTTTG
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #5
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
GCCCAAAACC CAAACCCAAA ACCTAG 2
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #6
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
AATTCGGGTT TGGGTTTGGG TTTGGG 26 ( 2 ) INFORMATION FOR SEQ ID NO: 41 :
( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 26 base pairs ( B ) TYPE : nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #6
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
GCCCAAACCC AAACCCAAAC CCCTAG 2
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #7
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
AATTCGGTTG GTTGGTTGGT TGGTTG 2
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #7
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GCCAACCAAC CAACCAACCA ACCTAG 2
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
AATTCGTGTG TGTGTGTGTG TGTGTG 2
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14A, TARGET SEQUENCE #8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
GCACACACAC ACACACACAC ACCTAG
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14B
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
TTTTTGGGGG TTTTTGGGGG
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 14B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
CCCCCAAAAA CCCCCAAAAA
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iϋ) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 15A
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
GGGGGTTTTT GGGGGTTTTT 2
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ϋi) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 15B, II
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 6..10
(D) OTHER INFORMATION: /note= "WHERE N IS INOSINE" ( ix) FEATURE :
(A) NAME/KEY: misc_feature
(B) LOCATION: 16..20
(D) OTHER INFORMATION: /note= "WHERE N IS INOSINE'
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
TTTTTNNNNN TTTTTNNNNN
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 15B, 12
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 6..10
(D) OTHER INFORMATION: /note= "WHERE N IS INOSINE"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 16..20
(D) OTHER INFORMATION: /note= "WHERE N IS INOSINE"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
CCCCCNNNNN CCCCCNNNNN 2
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 15B, Gl
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
TTTTTGGGGG TTTTTGGGGG 20
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: FIGURE 15B, Al
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
CCCCCAAAAA CCCCCAAAAA 20
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(C) INDIVIDUAL ISOLATE: FIGURE 15A
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: AAAAATTTTT AAAAATTTTT

Claims

IT IS CLAIMED:
1. An oligonucleotide capable of forming a triple-helix with a duplex nucleic acid molecule, which contains a target nucleic acid sequence, where said nucleic acid has first and second complementary strands and said first strand has a target sequence with at least one tract of purines adjacent at least one tract of pyrimidines, and said oligonucleotide contains at least one pyrimidine tract adjacent one purine tract, and where either
(i) the pyrimidine tract is parallel to the purine tract of the first strand target sequence and the purine tract is antiparallel to the purine tract of the second strand target sequence, or
(ii) the purine tract is antiparallel to the purine tract of the first strand target sequence and the pyrimidine tract is parallel to the purine tract of the second strand, and where the oligonucleotide pyrimidine and purine tracts have nucleic acid sequences which can pair, with the purine strands of the target sequence of the duplex, to form a triple helix.
2. The oligonucleotide of claim 1, wherein the target sequence of the duplex nucleic acid comprises
5'-{R1}i{Y2}j-3' 3'-{Y1>i{R2}J-5' where Rt and Y! are, respectively, complementary tracts of purines and pyrimidines, R2 and Y2 are, respectively, complementary tracts of purines and pyrimidines, and i and j are greater than or equal to 4, and where said oligonucleotide contains a sequence selected from the group consisting of 3'-
{ 1 },-{y2>r5 ' and s ' -CYihWr3 ' -- where r is A or T for R=A and r is G for R=G, and y is T when Y=A and y is C when Y=G.
3. The oligonucleotide of claim 1, wherein the target sequence of the duplex nucleic acid comprises
5'-{Y,}i{R2}j-3' 3'-{R,}i{Y2}j-5' where R! and Yj are, respectively, complementary tracts of purines and pyrimidines, R2 and Y2 are, respectively, complementary tracts of purines and pyrimidines, and i and j are greater than or equal to 4, and where said oligonucleotide contains a sequence selected from the group consisting of 5'- {r,}i y2}r ' and 3'-{y,}i{r2}j-5' , where r is A or T for R=A and r is G for R=G, and y is T when Y=A and y is C when Y=G.
4. The oligonucleotide of claim 1, wherein said target nucleic acid sequence contain one purine tract adjacent at least one pyrimidine tract.
5. The oligonucleotide of claim 1, wherein the target sequence contains at least about 8 nucleo¬ tides.
6. The oligonucleotide of claim 1, wherein the nucleic acid molecule is a duplex DNA molecule.
7. The oligonucleotide of claim 1, which further includes at least one moiety attached to the oligonucleotide, where the moiety is capable of cleaving the DNA molecule containing the target nucleic acid sequence.
8. The oligonucleotide of claim 7, wherein the cleaving moiety is selected from the group consist- ing of phenanthroline Cu(II), Zn(II), Fe(II)-EDTA, Cu(II)-bipyridine, and C (II)-terpyridine..
SUBSTITUTESHEET
9. A method of cleaving a duplex nucleic acid molecule, having first and second complementary strands, containing a target nucleic acid sequence, where said first strand has a target sequence with at least one tract of purines adjacent at least one tract of pyrimidines, comprising contacting the nucleic acid molecule with an oligonucleotide, where said oligonucleotide contains at least one pyrimidine tract adjacent one purine tract, and where either
(i) the pyrimidine tract is parallel to the purine tract of the first strand target sequence and the purine tract is antiparallel to the purine tract of the second strand target sequence, or (ii) the purine tract is antiparallel to the purine tract of the first strand target sequence and the pyrimidine tract is parallel to the purine tract of the second strand, and where the oligonucleotide pyrimidine and purine tracts have nucleic acid sequences which can pair, with the purine strands of the target sequence of the duplex, to form a triple helix, said oligonucleotide further includes at least one moiety which is capable of cleaving the nucleic acid molecule containing the target nucleic acid sequence, and where said contacting is at an oligonucleotide concentration effective to produce cleavage of the nucleic acid molecule containing the target nucleic acid sequence.
10. The method of claim 9, wherein said contacting further includes a reducing agent select¬ ed from the group consisting of mercaptopropionic acid and ascorbate.
SUBSTITUTE SHEET
11. The method of claim 9, wherein said moiety is selected from the group consisting of phenan¬ throline Cu(II), Zn(II), Fe(II)-EDTA, Cu(II)-bi- pyridine, and Cu(II)-terpyridine.
12. The method of claim 11, wherein cleaving moiety is phenanthroline.
13. The method of claim 12, wherein said contacting further includes the addition of CuS04 and mecaptopropionic acid.
14. The method of claim 9, wherein the nucleic acid molecule containing the target nucleic acid sequence is a duplex DNA molecule.
15. An oligonucleotide containing three regions capable of forming a triple-helix with a single-strand nucleic acid molecule, which contains a target nucleic acid sequence with at least one tract of purines adjacent at least one tract of pyrimidines, and said oligonucleotide contains (i) in the first region a nucleic acid sequence comple¬ mentary to the nucleic acid sequence of the target nucleic acid effective to form a duplex nucleic acid, (ii) in the second region, a stable abasic connecting sequence, and (iii) in the third region at least one pyrimidine tract in parallel to one purine tract on either strand of the duplex nucleic acid, adjacent at least one purine tract in anti¬ parallel to the purine tract on the opposite strand of the duplex nucleic acid, and where the oligonucleotide pyrimidine and purine tracts in the third region have nucleic acid sequences which can pair, with the purine strands of the target sequence of the duplex, to form a triple helix.
SUBSTITUTESHEET
16. The oligonucleotide of claim 15, wherein the nucleic acid molecule is a single stranded DNA molecule.
17. The oligonucleotide of claim 15, wherein the nucleic acid molecule is an RNA molecule.
18. The oligonucleotide of claim 15, which further includes at least one moiety attached to the oligonucleotide, where the moiety is capable of cleaving the nucleic acid molecule containing the target nucleic acid sequence.
19. A method of cleaving a single-strand nucleic acid molecule containing a target nucleic acid sequence, where said target sequence has at least one tract of purines adjacent at least one tract of pyrimidines, comprising contacting the nucleic acid molecule with an oligonucleotide, having three regions, comprised of (i) in the first region a nucleic acid sequence complementary to the nucleic acid sequence of the target nucleic acid effective to form a duplex nucleic acid, (ii) in the second region, a stable abasic connecting sequence, and (iii) in the third region at least one pyrimidine tract in parallel to one purine tract on either strand of the duplex nucleic acid, adjacent at least one purine tract in antiparallel to the purine tract on the opposite strand of the duplex nucleic acid, where the oligo¬ nucleotide pyrimidine and purine tracts in the third region have nucleic acid sequences which can pair, with the purine strands of the target sequence of the duplex, to form a triple helix, and where said oligonucleotide includes at least one moiety which is capable of cleaving the
SUBSTITUTESHEET nucleic acid molecule containing the target nucleic acid sequence, where said contacting is at an oligonucleotide concentration effective to produce cleavage of the nucleic acid molecule containing the target nucleic acid sequence.
20. The method of claim 19, wherein the nucleic acid molecule is a single stranded DNA molecule.
21. The method of claim 19, wherein the nucleic acid molecule is an RNA molecule.
22. The method of claim 21, wherein the RNA molecule is the HIV-1 genome.
23. The method of claim 22, wherein the oligonucleotide consists essentially of a sequence selected from the group of sequences presented as
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25, and SEQ ID NO:26.
SUBSTITUTESHEET
PCT/US1992/010792 1991-12-13 1992-12-11 Triple-helix formation at (punpyn).(punpyn) tracts WO1993012230A1 (en)

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US07/808,452 US6063612A (en) 1991-12-13 1991-12-13 Antiviral reagents based on RNA-binding proteins
US82693492A 1992-01-21 1992-01-21
US07/826,934 1992-01-21

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PCT/US1992/010770 WO1993012234A1 (en) 1991-12-13 1992-12-11 Antiviral reagents based on rna-binding proteins

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US6355450B1 (en) 1995-04-21 2002-03-12 Human Genome Sciences, Inc. Computer readable genomic sequence of Haemophilus influenzae Rd, fragments thereof, and uses thereof
US8183222B2 (en) 1995-06-06 2012-05-22 Trustees Of Boston University Method to inhibit cell growth using oligonucleotides
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