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WO1993005180A1 - Formation de complexes a triple helice d'adn double brin a l'aide d'oligomeres de nucleosides qui comprennent des analogues a base de purine - Google Patents

Formation de complexes a triple helice d'adn double brin a l'aide d'oligomeres de nucleosides qui comprennent des analogues a base de purine Download PDF

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Publication number
WO1993005180A1
WO1993005180A1 PCT/US1992/007246 US9207246W WO9305180A1 WO 1993005180 A1 WO1993005180 A1 WO 1993005180A1 US 9207246 W US9207246 W US 9207246W WO 9305180 A1 WO9305180 A1 WO 9305180A1
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oligomer
strand
nucleic acid
base
sequence
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PCT/US1992/007246
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English (en)
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Paul S. Miller
Purshotam Bhan
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The Johns Hopkins University
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Priority to EP92919326A priority Critical patent/EP0672171A4/fr
Priority to AU25544/92A priority patent/AU668886B2/en
Priority to JP5505283A priority patent/JPH06510203A/ja
Publication of WO1993005180A1 publication Critical patent/WO1993005180A1/fr
Priority to FI940922A priority patent/FI940922L/fi

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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • 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
    • 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
    • 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
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C12N2310/3125—Methylphosphonates
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/333—Modified A
    • 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
    • C12N2310/30—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
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/353—Nature of the modification linked to the nucleic acid via an atom other than carbon

Definitions

  • the present invention is directed to novel methods of detecting and locating specific sequences in double stranded DNA using nucleoside oligomers which are capable of specifically complexing with a selected double stranded DNA structure to give a triple helix structure.
  • Triple helical complexes containing cytosine and thymidine on the Hoogsteen (or third) strand have been found to be stable in acidic to neutral solutions, respectively, but have been found to dissociate on increasing pH. Incorporation of modified bases of T, such as 5-bromo-uracil and C, such as 5-methylcytosine, into the Hoogsteen strand has been found to increase stability of the triple helix over a higher pH range.
  • cytosine (C) In order for cytosine (C) to participate in the Hoogsteen-type pairing, a hydrogen must be available on the 3-N of the pyrimidine ring for hydrogen bonding. Accordingly, in some circumstances, C may be protonated at N-3.
  • DNA exhibits a wide range of polymorphic conformations, such conformations may be essential for biological processes. Modulation of signal transduction by sequence-specific protein-DNA binding and molecular interactions such as transcription, translation, and replication, are believed to be dependent upon DNA conformation.
  • Nuclease-resistant nonionic oligodeoxynucleotides consisting of a methylphosphonate (MP) backbone have been studied in vitro and in vivo as potential anticancer, antiviral and antibacterial agents.
  • ODN oligodeoxynucleotides
  • MP methylphosphonate
  • the 5'-3' linked internucleotide bonds of these analogs closely approximate the conformation of nucleic acid phosphodiester bonds.
  • the phosphate backbone is rendered neutral by methyl substitution of one anionic phosphoryl oxygen; decreasing inter- and intrastrand repulsion due to the charged phos phate groups.
  • Analogs with MP backbone can penetrate living cells and have been shown to inhibit mRNA translation in globin synthesis and vesicular stomatitis viral protein synthesis, and inhibit splicing of pre-mRNA in inhibition of HSV replication.
  • Mechanisms of action for inhibition by the nonionic analogs include formation of stable complexes with complementary RNA and/or DNA.
  • Nonionic oligonucleoside alkyl- and aryl-phosphonate analogs complementary to a selected single stranded foreign nucleic acid sequence can selectively inhibit the expression or function or expression of that particular nucleic acid without disturbing the function or expression of other nucleic acids present in the cell, by binding to or interfering with that nucleic acid.
  • U.S. Patent No. 4,469,863 and 4,511,713 See, e.g. U.S. Patent No. 4,469,863 and 4,511,713.
  • the use of complementary nuclease-resistant nonionic oligonucleoside methylphosphonates which are taken up by mammalian cells to inhibit viral protein synthesis in certain contexts, including Herpes simplex virus-1 is disclosed in U.S. Patent No. 4,757,055.
  • anti-sense oligonucleotides or phosphorothioate analogs complementary to a part of viral mRNA to interrupt the transcription and translation of viral mRNA into protein has been proposed.
  • the anti-sense constructs can bind to viral mRNA and were thought to obstruct the cells ribosomes from moving along the mRNA and thereby halting the translation of mRNA into protein, a process called "translation arrest” or "ribosomal-hybridization arrest.” (6)
  • Psoralen-derivatized oligonucleoside methylphosphonates have been reported capable of cross-linking either coding or non-coding single stranded DNA; however, double stranded DNA was not cross-linked.
  • the present invention is directed to methods of detecting or recognizing a specific segment of double stranded nucleic acid or double stranded nucleic acid sequence and to methods of preventing expression or function of a specific segment of double stranded nucleic acid having a given sequence. This is achieved without destroying the intactness of the duplex structure or interrupting the base-pairing of the double stranded nucleic acid.
  • the present invention is also directed to novel modified Oligomers which are useful for preventing expression and/or functioning of a selected double stranded nucleic acid sequence and which optionally include a nucleic acid modifying group.
  • the present invention is directed to novel Oligomers which comprise purine nucleoside analogs wherein the purine base has been chemically modified to favor the syn conformation.
  • the present invention is also directed to formation of a triple helix structure by the interaction of a specifie segment of double stranded nucleic acid and such an Oligomer wherein the Oligomer is sufficiently complementary to the nucleic acid segment to read it and base pair (or hybridize) thereto.
  • double stranded nucleic acids include double stranded DNA, as well as DNA:RNA hybrids and double stranded RNA.
  • the present invention is directed to methods of detecting or recognizing a specific segment of double stranded nucleic acid which comprises contacting said segment of nucleic acid with an Oligomer of the present invention which is sufficiently complementary to the sequence of purine bases in said segment of double stranded nucleic acid or a portion thereof to hydrogen bond (or hybridize) therewith thereby giving a triple helix structure.
  • the present invention is directed to methods of detecting or recognizing a specific segment of double stranded nucleic acid using an Oligomer which comprises at least one nucleosidyl unit which comprises a purine base modified to favor the syn conformation, preferably at the 8-position, and which is sufficiently complementary to said nucleic acid segment, or a portion thereof, to form a triple helix structure, wherein one strand of the duplex comprises a polypurine sequence of at least about 7 purine bases and wherein the Oligomer is parallel to the strand having the polypurine sequence.
  • an Oligomer which comprises at least one nucleosidyl unit which comprises a purine base modified to favor the syn conformation, preferably at the 8-position, and which is sufficiently complementary to said nucleic acid segment, or a portion thereof, to form a triple helix structure, wherein one strand of the duplex comprises a polypurine sequence of at least about 7 purine bases and wherein the Oligomer is parallel to the strand having the poly
  • guanine in the polypurine sequence is ready by a base in the Oligomer selected from 8-oxo-adenine and cytosine or a cytosine analog either of which is protonated at N-3 at Physiological pH and adenine in the polypurine sequence is read by a base in the Oligomer selected from 8-oxo-guanine, thymine and uracil.
  • the polypurine sequence may include up to about 50% pyrimidine bases.
  • cytosine in the polypurine sequence is read by a base in the Oligomer selected from 8-fluoroguanine, 8-fluoroadenine, 8-methoxyadenine and 8-azaadenine.
  • a base in the Oligomer selected from 8-fluoroguanine, 8-fluoroadenine, 8-methoxyadenine and 8-azaadenine.
  • the present invention is directed to methods of preventing or inhibiting expression or function of a specific segment of double stranded nucleic acid having a given sequence which comprises contacting said nucleic acid segment with an Oligomer of the present invention sufficiently complementary to said double stranded nucleic acid segment to form hydrogen bonds therewith, thereby giving a triple helix structure.
  • the invention is directed to methods of preventing or inhibiting expression or function of a specific segment of double stranded nucleic acid wherein one strand of the double stranded nucleic acid comprises a polypurine sequence of at least about 7 purine bases on one strand using an Oligomer which comprises at least one nucleosidyl unit which comprises a purine base modified to favor the syn conformation, preferably at the 8-position, and which Oligomer is parallel to the strand having the polypurine sequence.
  • guanine in the polypurine sequence is read by a base in the Oligomer selected from 8-oxo-adenine and cytosine or a cytosine analog either of which is protonated at N3 at physiological pH and adenine is read by a base in the Oligomer selected from 8-oxo- guanine, thymine and uracil.
  • the polypurine sequence may include up to about 50% pyrimidine bases.
  • cytosine in the polypurine sequence is read by a base in the Oligomer selected from 8-fluoroguanine, 8-methoxyguanine and 8-azaguanine
  • thymine or uracil in the polypurine sequence is read by a base in the Oligomer selected from 8-fluoroadenine, 8-methoxyadenine and 8-azaadenine.
  • the above noted 8-modifications of the purine base favor the syn conformation.
  • the present invention is directed to methods wherein the nucleic acid segment comprises a gene in a living cell and wherein formation of the triple helix structure permanently inhibits or inactivates said gene.
  • said Oligomer is modified to incorporate a nucleic acid modifying group which, after the Oligomer hydrogen bonds or hybridizes with the selected nucleic acid sequence, is caused to react chemically with the nucleic acid and irreversibly modify it.
  • modifications may include cross-linking Oligomer and nucleic acid by forming a covalent bond thereto, alkylating the nucleic acid, cleaving said nucleic acid at a specific location, or by degrading or destroying the nucleic acid.
  • third strand Oligomers comprise at least. one nucleosidyl unit having a purine base which is modified to favor the syn conformation.
  • These Oligomers comprise nucleosidyl units (or nucleoside monomers) which may be linked by any one of a variety of internucleosidyl linkages.
  • internucleosidyl linkages include, but are not limited to, phosphorus-containing linkages such as phosphodiester linkages, alkyl and aryl-phosphonate linkages, phosphoro- thioate linkages, phosphoramidite linkages and neutral phosphate ester linkages such as phosphotriester linkages; as well as internucleosidyl linkages which do not include phosphorus, such as morpholine linkages, formacetal linkages, sulfamate linkages, and carbamate linkages. Other internucleosidyl linkages known in the art may be used in these Oligomers.
  • these Oligomers may incorporate nucleosidyl units having modified sugar moieties which include ribosyl moieties, deoxyribosyl moieties and modified ribosyl moieties such as 2'-O-alkylribosyl (alkyl of 1 to 10 carbon atoms), 2'-O-arylribosyl, and 2'-halogen ribosyl, all optionally substituted with halogen, alkyl and aryl, and in particular, 2'-O-methylribosyl moieties.
  • modified sugar moieties which include ribosyl moieties, deoxyribosyl moieties and modified ribosyl moieties such as 2'-O-alkylribosyl (alkyl of 1 to 10 carbon atoms), 2'-O-arylribosyl, and 2'-halogen ribosyl, all optionally substituted with halogen, alkyl and
  • incorporation of nucleosidyl units having modified ribosyl, particularly 2'-O-methyl ribosyl, moieties may advantageously improve hybridization with the double stranded nucleic acid sequence and also improve resistance to enzymatic degradation.
  • the present invention provides novel nonionic alkyl- and aryl-phosphonate Oligomers comprising these purine nucleoside analogs which are sufficiently complementary to the purine sequence of a specific double stranded nucleic acid segment to hydrogen bond and form a triple helix structure.
  • Preferred are nonionic methylphosphonate Oligomers.
  • the present invention also provides Oligomers having nucleosidyl units in which a cytosine analog replaces cytosine and wherein said cytosine analog comprises a heterocycle which has a hydrogen available for hydrogen bonding at the ring position which corresponds to N-3 of cytosine and which is capable of forming two hydrogen bonds with a guanine base at neutral pH.
  • cytosine analogs include 5-methyl-cytosine, well as the analogs depicted in Table V.
  • the present invention is directed to Oligomers which comprise purine nucleoside analogs which can form a triplet with a purine-pyrimidine or pyrimidinepurine base pair, and to such Oligomers which can read a sequence on one strand of a duplex having both purine and pyrimidine nucleosides.
  • Oligomers are provided which comprise only purine nucleosides, including purine nucleoside analogs.
  • Oligomers are provided which comprise pyrimidine nucleosides in combination with these purine nucleoside analogs. These purine nucleoside analogs have been modified chemically, preferably at the 8-position, so that the syn conformation is favored.
  • nucleoside include 8-oxo- A, 8-oxo-G, 8-fluoro-A, 8-fluoro-G, 8-methoxy-A, 8- methoxy-G, 8-aza-A and 8-aza-G.
  • purine or “purine base” includes not only the naturally occurring adenine and guanine bases, but also modifications of those bases such as bases substituted at the 8-position.
  • the phosphonate group may exist in either an "R” or an "S” configuration.
  • Phosphonate groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
  • a "non-nucleoside monomeric unit” refers to a monomeric unit which does not significantly participate in hybridization of an Oligomer to a target sequence. Such monomeric units must not, for example, participate in any significant hydrogen bonding with a nucleoside, and would exclude monomeric units having as a component, one of the 5 nucleotide bases or analogs thereof.
  • oligonucleoside or “Oligomer” refers to a chain of nucleosides which are linked by internucleoside linkages which is generally from about 6 to about 100 nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually synthesized from nucleoside monomers, but may also be obtained by enzymatic means.
  • nucleoside/non-nucleoside polymers wherein one or more of the phosphorous group linkages between monomeric units has been replaced by a non- phosphorous linkage such as a, morpholino linkage, a formacetal linkage, a sulfamate linkage or a carbamate linkage.
  • alkyl- or aryl-phosphonate Oligomer refers to Oligomers having at least one alkyl- or aryl- phosphonate internucleosidyl linkage.
  • ''methylphosphonate Oligomer refers to Oligomers having at least one methylphosphonate internucleosidyl linkage.
  • neutral alkyl- or aryl- phosphonate Oligomer refers to neutral oligomers having neutral internucleosidyl linkages which comprise at beast one alkyl- or aryl- phosphonate linkage.
  • neutral methylphosphonate Oligomer refers to neutral Oligomers having internucleosidyl linkages which comprise at least one methylphosphonate linkage.
  • Third Strand Oligomer refers to Oligomers which are capable of reading a segment of a double stranded nucleic acid, such as a DNA duplex, and forming a triple helix structure therewith.
  • Complementary when referring to an Oligomer Third Strand refers to Oligomers having base sequences which hydrogen bond (and base pair or hybridize) with the purine base of a corresponding (Watson-Crick) base pair of a double stranded DNA to form a triple helix structure.
  • read refers to the ability of a nucleic acid residue to recognize through hydrogen bond interactions the base sequence of another nucleic acid.
  • a Third Strand Oligomer is able to recognize through hydrogen bond interactions the base pairs, in particular the purine bases, in the duplex of a segment of double stranded DNA.
  • triplet refers to a situation such as that depicted in Figures 1A, 1B; 2A to 2D and 7 to 10, wherein a base in the Third Strand has hydrogen bonded (and thus base paired) with a (Watson-Crick) base pair of a segment of double stranded DNA.
  • Fig. 1A and 1D depict triplets wherein a pyrimidine base in the Third Strand forms a triplet with a duplex DNA (Watson-Crick) base pair.
  • Fig. 2A to 2D depict triplets wherein a purine base in the Third strand forms a triplet with a duplex DNA (Watson-Crick) base pair.
  • Fig. 3A to 3B depict the base sequences of exemplary polypurine sequence triple helix structures wherein the Third Strand "reads" and, thus, base pairs with purine bases on one strand of a double stranded DNA.
  • Fig. 4A to 4E depict the base sequences of exemplary mixed sequence triple helix structures wherein the Third Strand "reads" and, thus base pairs with purine bases on both strands of a double stranded DNA.
  • Fig. 5 depicts a nucleosidyl unit having a modified sugar moiety with an alkyleneoxy link for lengthening internucleoside phosphorus linkages and processes for its preparation.
  • Fig. 6 depicts a nucleosidyl unit having a modified sugar moiety with an alkylene link for lengthening internucleoside phosphorus linkages and processes for its synthesis.
  • Fig. 7 depicts a triplet wherein 8-oxo-A in the third strand forms a triplet with the G of a G-C (Watson-Crick) base pair.
  • Fig. 8 depicts a triplet wherein 8-oxo-G in the Third Strand forms a triplet with the A of an A-T (Watson-Crick) base pair.
  • Fig. 9 depicts a triplet wherein 8-fluoro G in the Third Strand forms a triplet with the C of a C-G (Watson- Crick) base pair.
  • Fig. 10 depicts a triplet wherein 8-fluro A in the Third Strand forms a triplet with the T of a T-A (Watson- Crick) base pair.
  • Fig. 11 depicts CO spectra of triple helix structures
  • (- ) depicts a MP-Oligomer Third Strand
  • (---) depicts an Oligonucleotide Third Strand
  • Figs. 12A and 12B depict cross-linking of (A) single stranded DNA and (B) double stranded DNA using psoralenderivatized MP-Oligomers.
  • Fig. 13A depicts absorbance versus temperature profile of a solution of 0.5 ⁇ M I(OA) and 0.5 ⁇ M II ⁇ III (G-C).
  • Fig. 13AA depicts a continuous variation titration of I(OA) and II ⁇ III(G-C) at 1.5 ⁇ M total strand at 7°C
  • Fig. 13B depicts observed circular dichroism spectra of a solution containing 1.6 ⁇ M I(OA) and 1.6 ⁇ M II ⁇ III(G ⁇ C) at 10°C (--- ) and 35°C (••••) and the calculated weighted average spectra of 1.6 ⁇ M I(OA) and 1.6 ⁇ M II ⁇ III(G-C) at 10oc (----). All were performed in standard buffer.
  • the present invention involves the formation of triple helix structures with a selected double stranded DNA sequence by contacting said DNA with an Oligomer which is sufficiently complementary to the purine sequence of the double stranded DNA to form hydrogen bonds (or hybridize) therewith.
  • this invention includes the following aspects.
  • a first aspect concerns the reading (or recognition) of the base pairs in the double stranded DNA segment without opening the base pair, through the hydrogen bond formation by the bases in the Third Strand with the extra hydrogen bond sites of purines in the double stranded DNA, such as adenine and guanine.
  • the reading of the base pair sequence in the DNA duplex is always done by reading the purine of the base pair, through hydrogen bond formation with the bases in the Third strand, with the remaining available hydrogen bonding sites of the purines in the DNA.
  • Either purines (such as adenine (A) or guanine (G)) or pyrimidines (thymine (T) or cytosine (C)) in the Third Strand can form hydrogen bonds with the purines in the DNA, i.e.:
  • Adenine (A) in the base pair of DNA can be read or hydrogen bonded with A or T in the Third Strand, or in a preferred aspect, A is read by 8-oxo-G.
  • the phosphorus-containing backbone of the Third Strand comprises methylphosphonate groups as well as naturally occurring phosphodiester groups.
  • a second aspect of this invention is to read the purine (A or G) in the double stranded DNA totally by the use of purine bases from the Third Strand.
  • the polarity of the strands, either the anti-parallel or parallel direction of the Third Strand in respect to the strand containing the purine to be read in the duplex is important.
  • the conformational state of the nucleoside is defined principally by the rotation of these two more or less rigid planes, i.e. the base and the pentose, relative to each other about the axis of the C'-1 to N-9 or N-1 bond.
  • the sugar-base torsion angle, ⁇ CN has been defined as "the angle formed by the trace of the plane of the base with the projection of the C-1' to 0-1' bond of the furanose ring when viewed along the C'-1 to a bond.
  • This angle will be taken as zero when the furanose-ring oxygen is antiplanar to C-2 of the pyrimidine or purine ring and positive angles will be taken as those measured in a clockwise direction when viewing C-1' to N.”
  • This angle has also been termed the glycosyl torsion angle.
  • ⁇ CN glycosyl torsion angle
  • the conformations of the purine nucleosidyl units in the Third Strand are influenced by the polarity (parallel (5' to 3') or anti- parallel (3' to 5') direction) of the strand containing the purine bases to be read in the DNA in relation to the Third Strand.
  • the conformation of the purine nucleosidyl unit in the Third Strand should be in the conformation.
  • the conformation of the purine nucleosidyl unit in the Third Strand in reading the corresponding purine in the anti-parallel strand in the duplex should exist in anti conformation.
  • a Third Strand which has the same polarity as (i.e. is parallel to) either one strand or the opposite strand .
  • DNA duplex having the same polarity of the Watson strand from 5' to 3' would be as follows:
  • the purine nucleosidyl units (A or G) of the Third Strand need to be in syn conformation in reading the purines in the base pair (A or G) of the parallel ("Watson") strand of the double stranded DNA.
  • the purine nucleosidyl unit needs to be in anti conformation.
  • adenine in the Third Strand is used to read adenine in the duplex and guanine in the Third Strand is used to read guanine in the duplex.
  • the length of the link between the 5' carbon of the "nucleosidyl unit one" to the 3' oxygen of the subsequent "nucleosidyl unit two” may be increased by two atoms (such as -CH 2 CH 2 -) or by 3 atoms (such as -O-CH 2 -CH 2 -), thereby lengthening the linkage between individual nucleosidyl units by 2 to 6 ⁇ .
  • separation of the units be increased by a number of atoms ranging from 1 to 6.
  • Figure 6 illustrates an example of a nucleosidyl unit comprising this lengthening link format and proposed synthetic routes.
  • a second lengthening link format for the phosphorus backbone would comprise non-uniform lengthening links.
  • Links having internucleosidyl distances on the order of the standard phosphodiester backbone for the Third Strand would be employed when the purines being read were on the same strand, while a lengthened link (15-17 ⁇ in length) which could comprise lengthening links on the 3' carbon of one nucleosidyl unit and on the 5'- carbon of its neighbor, would be employed to read the purine bases located on opposite strands.
  • Such a non-uniform lengthening link format would be particularly suitable for use in Third Strands comprising both pyrimidine (or pyrimidine analog) and purine bases.
  • Third Strand Oligomers which can read both purine and pyrimidine bases, by hydrogen bond formation with the bases in the Third Strand with available hydrogen bonding sites of both purine and pyrimidine bases in the double stranded nucleic acid.
  • This allows ttiie Oligomer to read a sequence such as one where a homopurine run is interrupted by one or more pyrimidines bases without necessitating use of a lengthening link on the Third Strand Oligomer to allow for switching strands which are read at a site of a pyrimidine interruption, so that the purine on the other strand could be read to give the triplex.
  • Use of these Oligomers is advantageous in that only one strand need be read. Reading of only one strand also simplifies synthesis of the Third Strand Oligomers.
  • Oligomers are provided wherein 8-oxo-adenine is used to complex with (or read) G, 8-oxo-guanosine is used to complex with (or read) A, 8-fluoro G is used to read C and 8-fluoro A is used to read T (or U?).
  • 8-oxo-adenine is used to complex with (or read) G
  • 8-oxo-guanosine is used to complex with (or read) A
  • 8-fluoro G is used to read C
  • 8-fluoro A is used to read T (or U?).
  • triplets are formed wherein a pyrimidine base in the Third Strand forms hydrogen bonds and, thus base pairs with the purine base of a base pair of the double stranded DNA sequence. Examples of such triplets where having a pyrimidine base as the base in the Third Strand are shown in Figures 1A and 1B.
  • Fig. 1A depicts a triplet having T as the Third Strand base which forms hydrogen bonds and, thus, base pairs with the A of the double stranded DNA.
  • the T of the Third Strand is aligned parallel to the A-containing strand of the double stranded DNA, and anti-parallel to the T-containing strand of the double stranded DNA (which is also anti-parallel to the A-containing strand). Accordingly, the sequence for that triplet is written as follows:
  • Fig. 1B depicts a triplet having a protonated cytosine (C+) as the Third Strand base which forms hydrogen bonds and, thus, base pairs with a G of a G-C base pair in the double stranded DNA.
  • the cytosine base in the Third Strand must be protonated at N3 in order to form hydrogen bonds necessary for a stable triplet.
  • a cytosine may be replaced with a cytosine analog having a guaternary nitrogen at a position analogous to N3.
  • the C+ (or its analog) of the Third Strand is aligned parallel to the G-containing strand of the double stranded DNA, and anti-parallel to the C-containing strand of the double stranded DNA (which is also anti-parallel to the G-containing strand). Accordingly, such a triplet sequence is written as follows:
  • triplets are formed wherein a purine base in the Third Strand forms hydrogen bonds with and, thus, base pairs with the same purine base in one strand of the double stranded DNA.
  • the purine base in the Third Strand is capable of base pairing with the purine base of double stranded DNA such that the strand polarity of the Third Strand containing the purine base may be aligned either parallel or anti- parallel to strand polarity of the strand containing the purine to be read in the double stranded DNA.
  • Fig. 2A depicts a triplet where the A of the Third Strand is aligned parallel to the A of the double stranded DNA, and anti-parallel to the T of the double stranded DNA.
  • the glycosyl (C-N) torsion angle of the Third Strand A is in the syn conformation, and the glycosyl torsion angles of the A and T bases of the double stranded DNA are both in the anti conformation.
  • Fig. 2B depicts a triplet where the A of the Third Strand is aligned anti-parallel to the A of the double stranded DNA and parallel to the T of the double stranded DNA.
  • the glycosyl torsion angles of all three bases are in the anti conformation.
  • Fig. 2C depicts a triplet wherein the G of the Third Strand is aligned parallel to the G of the double stranded DNA, and anti-parallel to the C of the double stranded DNA.
  • the glycosyl torsion angle of the Third strand G is in the syn conformation
  • the glycosyl torsion angles of the G-C bases of the double stranded DNA are both in the anti conformation.
  • Fig. 2D depicts a triplet wherein the G of the Third strand is aligned anti-parallel to the G of the double stranded DNA and parallel to the C of the double stranded DNA. In such circumstance, the glycosyl torsion angles for all three bases are in the anti conformation.
  • Such a triplet sequence is written as follows:
  • triplets are formed using a Third Strand Oligomer which comprises a nucleoside which comprises a purine analog which has been chemically modified to favor the base being in the syn configuration.
  • the Third strand Oligomer is parallel to the strand of the double-stranded nucleic acid being read.
  • Figure 8 depicts a triplet wherein an 8-oxo-G of the third strand is aligned parallel to A of the double stranded nucleic acid and antiparallel to the T or U of the double stranded nucleic acid.
  • the glycosyl (C-N) torsion angle of the third strand 8- oxo-G is in the syn configuration and the glycosyl torsion angles of the A and T or U bases of the double stranded nucleic acid are both in the anti-conformation.
  • G is in the syn conformation and the glycosyl torsion angles of the C and G bases of the double-stranded nucleic acid are both in the anti-conformation.
  • Figure 10 depicts a triplet wherein an 8-fluoro-A in the third strand is aligned parallel to the T (or U) of a double stranded nucleic acid.
  • the glycosyl (C-N) torsion angle of the third strand 8-fluoro- A is in the syn conformation and the glycosyl torsion angles of the A and T (or U) bases of the double stranded nucleic acid are both in the anti-conformation.
  • Mixed sequences are sequences wherein the purine bases of the selected nucleic acid sequence are located on both strands of the double stranded nucleic acid.
  • the polypurine sequence on one strand of the double-stranded nucleic acid sequence may comprise up to about 50% pyrimidine bases.
  • the Third Strand Oligomer must be able to hydrogen bond and, thus, base pair with the purine base of each base pair of the double stranded nucleic acid sequence without regard to which strand of the double stranded nucleic acid the purine base is located on. Accordingly, the Third Strand Oligomer must be able to "read” across the double stranded nucleic acid. Examples of mixed sequences including an appropriately complementary Third Strand are depicted in Fig. 4A to 4E.
  • Fig. 4A depicts a mixed sequence having a pyrimidine- rich Third Strand.
  • Fig. 4B depicts a mixed sequence having a purine-rich Third Strand.
  • Fig. 4C, 4D and 4E depict mixed sequences having Third Strands containing only purine bases.
  • S denotes syn and "a” denotes anti; no superscript denotes anti.
  • the Third Strand Oligomer must "read” across the double strand from one strand to the opposite strand in order to base pair with the purine base, it may be advantageous to provide a lengthened internucleosidyl phosphorus linkage by incorporation of the previously- described backbone link formats into the phosphorus backbone which connects the sugar moieties of the nucleosidyl units.
  • a mixed sequence comprises a one or more polypurine sequences which include up to about 50% pyrimidine bases on one strand of the double stranded nucleic acid sequence
  • both purine and pyrimidine bases of the sequence may be read.
  • the pyrimidine bases are read using the following modified purine bases.
  • Oligomers having at least about 7 nucleosides which is usually a sufficient number to allow for specific binding to a desired purine sequence of a segment of double stranded nucleic acid. More preferred are Oligomers having from about 8 to about 40 nucleotides; especially preferred are Oligomers having from about 10 to about 25 nucleosides. Due to a combination of ease of synthesis, with specificity for a selected sequence, coupled with minimization of intra-Oligomer, internucleoside interactions such as folding and coiling, it is believed that Oligomers having from about 14 to about 18 nucleosides comprise a particularly preferred group . (1) Preferred Oligomers
  • oligomers may comprise either ribosyl moieties or deoxyribosyl moieties or modifications thereof, such as 2'-O-alkyl ribosyl moieties.
  • nucleotide Oligomers i. e., having the phosphodiester internucleoside linkages present in natural nucleotide Oligomers, as well as other oligonucleotide analogs
  • Oligomers having other internucleasidyl linkages such as Oligomers which comprise oligonucleoside alkyl and arylphosphonate analogs, phosphorothioate oligonucleoside analogs, phosphoroamidate analogs and neutral phosphate ester oligonucleotide analogs.
  • oligonucleoside alkyl- and aryl- phosphonate analogs in which phosphonate linkages replace one or more of the phosphodiester linkages which connect two nucleosidyl units are included, as well as Oligomers having non-phosphorous linkages.
  • Oligomers having non-phosphorous linkages The preparation of some such oligonucleosidyl alkyl and arylphosphonate analogs and their use to inhibit expression of preselected single stranded nucleic acid sequences is disclosed in U.S. Patent Nos. 4,469,863; 4,511,713; 4,757,055; 4,507,433; and 4,591,614, the disclosures of which are incorporated herein by reference.
  • One particularly preferred class of these phosphonate analogs are methylphosphonate Oligomers.
  • MP-Oligomers Synthetic methods for methylphosphate Oligomers are described in Lee, B.L. et al. Biochemistry 27:319-3203 (1988) and Miller, P.S., et al., Biochemistry 25:5092-5097 (1986), the disclosures of which are incorporated herein by reference.
  • MP-Oligomers should be more resistant to hydrolysis by various nuclease and esterase activities, since the methylphosphonyl group is not found in naturally occurring nucleic acid molecules. Due to the nonionic nature of the methylphosphonate linkage, these MP-oligomers should be better able to cross cell membranes and thus be taken up by cells.
  • MP-Oligomers are more resistant to nuclease hydrolysis, are taken up in intact form by mammalian cells in culture and can exert specific inhibitory effects on cellular DNA and protein synthesis (See, e.g., U.S. Patent No, 4,469,863).
  • MP-Oligomers having at least about seven nucleosidyl units, more preferably at least about 8, which is usually sufficient to allow for specific recognition of the desired segment of double stranded DNA. More preferred are MP-Oligomers having from about 8 to about 40 nucleosides, especially preferred are those having from about 10 to about 25 nucleosides. Due to a combination of ease of preparation, with specificity for a selected sequence and minimization of intra-Oligomer, internucleoside interactions such as folding and coiling, particularly preferred are MP-Oligomers of from about 14 to 18 nucleosides.
  • the selected double stranded nucleic acid sequences are sequenced and MP-Oligomers complementary to the purine sequence are prepared by the methods disclosed in the above noted patents and disclosed herein.
  • preferred Oligomers include those comprising nucleosidyl units having purine bases modified to favor the syn conformation. These bases include those modified at the 8-position such as 8-oxo-A, 8-oxo-G, 8-fluoro-A, 8-fluoro-G, 8-methoxy-A, 8-methoxy-G, 8-aza-A 8-aza-G, and other similarly modified purines known in the art.
  • Oligomers are useful in determining the presence or absence of a selected double stranded nucleic acid sequence in a mixture of nucleic acids or in samples including isolated cells, tissue samples or bodily fluids.
  • These Oligomers are useful as hybridization assay probes and may be used in detection assays. When used as probes, these Oligomers may also be used in diagnostic kits.
  • labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, intercalating agents such as acridine, or groups capable of cleaving the targeted portion of the viral nucleic acid such as molecular scissors like o-phenanthrolinecopper or EDTA-iron may be incorporated in the MP-Oligomers.
  • Oligomers may be labelled by any of several well known methods.
  • Useful labels include radioisotopes as well as nonradioactive reporting groups.
  • Isotopic labels include 3 H, 35 S, 32 P, 125 I, Cobalt and 14 C.
  • Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, end labelling, second strand synthesis, and reverse transcription.
  • hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radioisotope used for labelling.
  • Non-isotopic materials can also be used for labelling, and may be introduced by the incorporation of modified nucleosides or nucleoside analogs through the use of enzymes or by chemical modification of the Oligomer, for example, by the use of non-nucleotide linker groups.
  • Non-isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands.
  • One preferred labelling method includes incorporation of acridinium esters.
  • Such labelled Oligomers are particularly suited as hybridization assay probes and for use in hybridization assays.
  • these Oligomers When used to prevent function or expression of a double stranded nucleic acid sequence, these Oligomers may be advantageously derivatized or modified to incorporate a nucleic acid modifying group which may be caused to react with said nucleic acid and irreversibly modify its structure, thereby rendering it non-functional.
  • a nucleic acid modifying group which may be caused to react with said nucleic acid and irreversibly modify its structure, thereby rendering it non-functional.
  • Our co-pending patent application U.S. Serial No. 924,234, filed October 28, 1986, the disclosure of which is incorporated herein by reference, teaches the derivatization of Oligomers which comprise oligonucleoside alkyl and arylphosphonates and the use of such derivatized oligonucleoside alkyl and arylphosphonates to render targeted single stranded nucleic acid sequences nonfunctional.
  • nucleic acid modifying groups may be used to derivatize these Oligomers.
  • Nucleic acid modifying groups include groups which, after the derivatized Oligomer forms a triple helix structure with the double stranded nucleic acid segment, may be caused to form a covalent linkage, cross-link, alkylate, cleave, degrade, or otherwise inactivate or destroy the nucleic acid segment or a target sequence portion thereof, and thereby irreversibly inhibit the function and/or expression of that nucleic segment.
  • the location of the nucleic acid modifying groups in the Oligomer may be varied and may depend on the particular nucleic acid modifying group employed and the targeted double stranded nucleic acid segment. Accordingly, the nucleic acid modifying group may be positioned at the end of the Oligomer or intermediate the ends. A plurality of nucleic acid modifying groups may be included.
  • the nucleic acid modifying group is photoreactable (e. g., activated by a particular wavelength, or range of wavelengths of light), so as to cause reaction and, thus, cross-linking between the
  • Oligomer and the double stranded nucleic acid are referred to as Oligomer and the double stranded nucleic acid.
  • nucleic acid modifying groups which may cause cross-linking are the psoralens, such as an aminomethyltrimethyl psoralen group (AMT).
  • AMT aminomethyltrimethyl psoralen group
  • the AMT is advantageously photoreactable, and thus must be activated by exposure to particular wavelength light before cross-linking is effectuated.
  • Other cross-linking groups which may or may not be photoreactable may be used to derivatize these Oligomers.
  • the DNA modifying groups may comprise an alkylating agent group which, on reaction, separates from the Oligomer and is covalently bonded to the DNA segment to render it inactive.
  • alkylating agent groups are known in the chemical arts and include groups derived from alkyl halides, haloacetamides, phosphotriesters and the like.
  • DNA modifying groups which may be caused to cleave the DNA segment include transition metal chelating complexes such as ethylene diamine tetraacetate (EDTA) or a derivative thereof, other groups which may be used to effect cleaving include phenanthroline, porphyrin or bleomycin, and the like.
  • EDTA ethylene diamine tetraacetate
  • iron may be advantageously tethered to the Oligomer to help generate the cleaving radicals.
  • EDTA is a preferred DNA cleaving group
  • other nitrogen containing materials such as azo compounds or nitreens or other transition metal chelating complexes may be used.
  • the nucleosidyl units of Third Strand Oligomers which read purine bases on both strands of a double stranded DNA sequence may comprise a mixture of purine and pyrimidine bases or only purine bases. Where purine bases on both strands of a double stranded DNA sequence are to be read, it is preferred to use Oligomers having only purine bases.
  • purine-only Oligomers are advantageous for several reasons: (a) purines have higher stacking properties than pyrimidines, which would tend to increase stability of the triple helix structure; (b) use of purines only eliminates the need for either protonation of cytosine (so it has an available hydrogen for hydrogen bonding at the N-3 position at neutral pH) or use of a cytosine analog having such an available hydrogen at the position which corresponds to N3 on the pyrimidine ring; and allows use of a universal lengthening link.
  • the purine bases are normally in the anti conformation; however, the barrier for a base to roll over to the syn conformation is low.
  • the purines on the Third Strand may assume the syn conformation during the hydrogen bonding process.
  • the purine may be modified at the 8-position with a substitutent such as methyl, bromo, isopropyl or other bulky group so it will assume the syn configuration under normal conditions. Nucleosidyl units comprising such substituted purines would thus normally assume the syn conformation.
  • the present invention contemplates the optional incorporation of such 8-substituted purines in place of unsubstituted A or G. Studies with our (Kendrew) models indicate that such substitutions should not affect formation of the triple helix structure.
  • a Third Strand Oligomer comprising both pyrimides and purines is used to read purines on both strands of a double stranded DNA
  • a non-uniform link format is used as described herein to allow the Third Strand to read across from one strand of the duplex to the other.
  • novel Oligomers comprise nucleosidyl units wherein cytosine has been replaced by a cytosine analog comprising a heterocycle which has an available hydrogen at the ring position analogous to the 3-N of the cytosine ring and is capable of forming two hydrogen bonds with a guanine base at neutral pH and thus does not require protonation as does cytosine for Hoogstein-type base pairing, or formation of a triplet.
  • Suitable nucleosidyl units comprise analogs having a six-membered heterocyclic ring which has a hydrogen available for hydrogen bonding at the ring position corresponding to N-3 of cytosine and which is capable of forming two hydrogen bonds with a guanine base at neutral pH and include 2'-deoxy-5,6-dihydro-5-azadeoxycytidine
  • Oligomers are provided which comprise nucleosidyl units which comprise a purine base or analog which has been modified at the 8-position to favor the syn conformation.
  • these purine analogs include 8-oxo-A which has been demonstrated to form a triplet with a G ⁇ C base pair (see Example 4).
  • the purine analog 8-oxo-G may be used to read the A of an A ⁇ T base pair.
  • Certain 8-modified purine analogs may be used to read the pyrimidine base of a (Watson-Crick) base pair.
  • the 8-fluoro-A may be used to read T and 8- fluoro G may be used to read C.
  • the 8-methoxy and 8-aza analogs of a and G may be used to read T and C respectively.
  • Oligomers comprising nucleosidyl units which comprise modified sugar moieties having lengthening links (see Figures 5 and 6) may be conveniently prepared by these methods.
  • Oligomers comprising phosphodiester internucleosidyl phosphorus linkages may be synthesized using any of several conventional methods, including automated solid phase chemical synthesis using cyanoethylphosphoroamidite precursors (29).
  • nucleosidyl units comprising cytosine analogs (see Table 5) may be incorporated into the MP-Oligomer by substituting the appropriate cytidine analog (see Table 5) in the reaction mixture.
  • MP-Oligomers may be prepared using modified nucleosides where either the bond between the 5'-carbon and the 5 '-hydroxyl or the 3'-carbon and the 3'-hydroxyl off the sugar moiety has been substituted with a alkyleneoxy group, such as ethyleneoxy group.
  • Figure 5 shows proposed reaction schemes for preparation of intermediates for modified nucleosides having either a 3'-(ethyleneoxy) or 5'-(ethyleneoxy) link.
  • B represents a base
  • Tr and R represent protecting groups
  • nucleosidyl units having such lengthening links at both the 3'- and 5'- positions of the sugar moiety may be prepared.
  • MP-Oligomers having a slightly lengthened internucleoside link on the phosphorus backbone.
  • Such MP-Oligomers may be prepared using nucleosides in which the sugar (deoxyribosyl or ribosyl) moiety has been modified to replace the 5'-hydroxy with a ⁇ -hydroxyethyl (HO-CH 2 CH 2 -) group synthetic schemes for the preparation of such a 5'- ⁇ -hydroxyethyl-substituted nucleosides is depicted in Figure 6.
  • Figure 6 depicts a proposed reaction scheme for a 5'- ⁇ -hydroxyethyl-substituted sugar analog.
  • DCC denotes dicyclohexylcarbodiimide
  • DMSO denotes dimethylsulfoxide.
  • B is a base.
  • Suitable protecting groups, R include t-butyldimethyl silyl and tetrahydropyranyl.
  • MP-oligomers incorporating the above-described modified nucleosidyl units are prepared as described above, substituting the modified nucleosidyl unit.
  • nucleosidyl units having the same lengthening links may be employed.
  • a mixture of nucleosidyl units having no lengthening link and lengthening links are used; nucleosidyl units having lengthening links at both the 3'- carbon and the 5'-carbon of the sugar moiety may be advantageous.
  • Derivatized Oligomers may be readily prepared by adding the desired DNA modifying groups to the Oligomer.
  • the number of nucleosidyl units in the Oligomer and the position of the DNA modifying group(s) in the Oligomer may be varied.
  • the DNA modifying group(s) may be positioned in the Oligomer where it will most effectively modify the target sequence of the DNA. Accordingly, the positioning of the DNA modifying group may depend, in large measure, on the DNA segment involved and its key target site or sites, although such optimum position can be readily determined by conventional techniques known to those skilled in the art.
  • a specific segment of double stranded DNA may be detected or recognized using an MP-Oligomer Third Strand which reads the purine bases of the duplex of the double-stranded nucleic acid according to the triplet base pairing guidelines described herein.
  • the MP-Oligomer Third Strand has a sequence selected such that the base of each nucleosidyl unit will form a triplet with a corresponding base pair of the double stranded nucleic acid DNA to give a triple helix structure.
  • Detectably labeled Oligomers may be used as probes for use in hybridization assays, for example, to detect the presence of a particular double- stranded nucleic acid sequence.
  • the present invention also provides a method of preventing expression or function of a selected sequence in double stranded nucleic acid by use of an MP-Oligomer which reads the nucleic acid sequence and forms a triple helix structure. Formation of the triple helix may prevent expression and/or function by modes such as preventing opening of the duplex for transcription, preventing of binding of effector molecules (such as proteins), etc.
  • Derivatized Oligomers may be used to detect or locate and then irreversibly modify at target site in the nucleic acid duplex by cross-linking (psoralens) or cleaving one or both strands (EDTA). By careful selection of a target site for cleavage, the Oligomer may be used as a molecular scissors to specifically excise a selected nucleic acid sequence.
  • the Oligomers may be derivatized to incorporate a nucleic acid reacting or modifying group which can be caused to react with the segment or a target sequence thereof to irreversibly modify, degrade or destroy the nucleic acid and thus irreversibly inhibit its functions.
  • these Oligomers may be used to irreversibly inactivate or inhibit a particular gene or target sequence of the game in a living cell, allowing selective inactivation or inhibition. These Oligomers could then be used to permanently inactivate, turn off or destroy genes which produced defective or undesired products or if activated caused undesirable effects.
  • kits for detecting a particular double stranded nucleic acid sequence which comprises a detectably labeled purine MP-Oligomer Third Strand selected to be able sufficiently complementary to the purine sequence to be able to read the sequence and form a triple helix structure.
  • Triple-stranded poly(dT 10 )-poly-(dA 10 )-poly(dT 10 ) [T 1 AT 2 ) coordinates were obtained from the A-DNA x-ray structure of Arnott and Seising (10). The same coordinates were used for the starting geometry of poly(dT 10 )-poly (dA 10 )- poly(dT 10 ) methylphosphonate [T 1 AT 2 MP] .
  • Geometry optimization and partial atomic charge assignments for the dimethyl ester methylphosphonate fragment were calculated by ab initio quantum mechanical methods with QUEST (version 1.1) using 3-21G* and STOG* basis sets, respectively (11). The latter basis set was used to maintain uniform charge assignments with those previously calculated for nucleic acids in the AMBER database.
  • the final monopole atomic charge assignments for the MP fragment were made to obtain a neutral net charge for each base, furanose, and MP backbone of the third DNA strand.
  • Alternating R P_ and S P_ methyl substitution of the backbone phosphoryl oxygens of the T 2 MP strand was done by stereo computer graphics.
  • the substitution of MP diastereomers was made in this manner to approximate experimental yield, since the synthesis cannot be controlled.
  • Molecular mechanics and molecular dynamics calculations were made with a fully vectorized version of AMBER (version 3.1), using an all-atom force field (12, 13). All calculations were performed on CRAY X-MP/24 and VAX 8600 computers.
  • the negative charge of the DNA phosphate backbone was rendered neutral by placement of positive counterions within 4 A of the phosphorus atoms bisecting the phosphate oxygens; counterions were not placed on the MP-substituted strand.
  • the triple helices and counterions were surrounded by a 10A shell of TIP3P water (14) molecules with periodic boundary conditions.
  • TIP3P water 14
  • the box dimensions were 101, 686.8 A 3 for T 1 AT 2 , and 124,321.1 A 3 for T 1 AT 2 MP.
  • the third DNA strand with MP backbone resulted in several changes consistent with enhanced binding of the ODN with the MP backbone in the triple helix.
  • the average hydrogen bond distances and mean atomic fluctuations are consistently smaller in the T 1 AT 2 MP triplet (Table 1).
  • the interstrand phosphorus atoms distance was 9.6A (+/-0.91) for A-T 2 and 8.3A (+/-0.58) for A-T 2 MP.
  • the reduced interstrand phosphorus atom distance and smaller mean atomic fluctuations between the second and Third Strands are due to decreased interstrand electrostatic repulsion accompanying MP substitution in the backbone.
  • the sugar puckering pattern of the MP substituted helix had a greater proportion of 01'endo and C2'endo conformations in contrast to the unsubstituted helix.
  • Analysis of other conformational parameters support the hybrid conformational nature of these triple helices.
  • the helical twist angle (between T1 and A strands) averaged 39.4 degrees (+ ⁇ -2.86) for the T 1 AT 2 structure and is more consistent with a B-DNA conformation (range 36-45).
  • the T 1 AT 2 MP helical twist angle averages 32.0 degrees (+ ⁇ -2.19) and is closer to that of A-DNA (range 30-32.7).
  • the average, helical repeat singles (between the T1 and A strands) for the entire structure are for 10.2 T 1 AT 2 and 11.2 degrees for T 1 AT 2 MP.
  • the average intrastrand phosphorus atom distances over the 40 psec trajectory are presented in Table 3.
  • the intrastrand phosphorus distances of the T 1 strands are most consistent with an A-DNA conformation (7.0 A).
  • the interstrand phosphorus distances of the T 2 MP strand are more consistent with a B-DNA conformation, in contrast to values more consistent with A-DNA for the T 2 strand.
  • the large perturbation of the ⁇ dihedral and variable conformational fluctuation of the R p- and S p- MP diastereoisomers in the triplet are due to nonbonded and hydrophobic interactions.
  • the Sp-MP groups are in close proximity to thymine methyl groups (in the major groove) on the same DNA strand, and interact by Van der Waals forces, which could locally destabilize the helix by "locking" the thymine to the Sp-MP backbone.
  • There are greater deviations in the ⁇ dihedral of the Rp-MP groups since these groups project out into the solvent water and are positioned farther away from the thymine methyl groups.
  • Circular dichroism spectroscopy studies were performed using Triple Helix Structures formed using a combination following nucleoside oligomers.
  • Figure 11 shows the CD spectra for triple helix (a)
  • Psoralen derivatized dTp(T) 6 oligomers were prepared as described in Lee, B.L., et al., Biochemistry 27:3197-3203 (1988).
  • the T 7 oligomers were allowed to hybridize with DNA having the following sequence including a 15-mer poly A subsequence:
  • MP-oligomers derivatized with 4 ' - (aminoethyl) amino- methyl-4, 5 ' ,8-trianethyl-psoralen [" (ae)AMT"] , 4 ' - (aminobutyl) -aminomethyl-4 , 5 ' , 8-trimethylpsoralen
  • Figure 12B shows crosslinking of the double stranded DNA with the double stranded DNA sequence.
  • I ⁇ II ⁇ III (OA ⁇ G ⁇ C) was further confirmed by the circular dichoism (CD) spectra shown in Figure 13B.
  • CD circular dichoism
  • the CD spectrum of I(OA) ⁇ II ⁇ III (G ⁇ C) showed DeltaE at 220 nm and 280 nm, behavior which has been identified as characteristic of triplex formation. (See Pilch, D.S., et al., Proc. Natl. Acad. Sci. (USA) 87:1942-1946 (1990)).
  • a temperature above the Tm of the Third Strand the CD spectrum was essentially identical to the sum of the spectra of I(OA) and II ⁇ III (G ⁇ C).
  • CD spectra of the triplex I ⁇ II ⁇ III behaved in a similar manner at 10oC and 40oC, and the spectrum of that triplex was virtually identical to that of I ⁇ II ⁇ III (OA ⁇ G ⁇ C).
  • 8-oxoadenosine can form two hydrogen bonds with G (at the N-7 and 0-6 atoms of G).
  • 8-oxo-adenosine has been shown to exist in the keto form with the base in the syn configuration. Triplex formation by 1(OA) with ll ⁇ lll (G ⁇ C) could occur if the keto form of 8-oxoadenine was in the syn conformation.
  • the proposed hydrogen bonding scheme was supported by the similarities in melting behavior of I ⁇ II ⁇ III (OA ⁇ G ⁇ C) and I ⁇ II ⁇ III (C ⁇ G ⁇ C) as noted in Table VI.
  • the increased Tm may be due to formation of a single hydrogen bond between the N-6 exocyclic amino group of 8-oxoadenine and the 0-4 of the uracil in the u.A base pair.
  • a single hydrogen bond could also be formed by 8-oxoadenine and the 0-4 of thymine, no triplex formation was observed for a 1:1 mixture of I(OA) and II ⁇ III (T ⁇ A). In that instance, triplex formation may be prevented by the interaction of the 5-methyl group of thyamine with the 5-membered ring of 8-oxo-adenine.
  • Q Averages of sugar pucker (Q), phase, and conformational populations of furanose for T 1 AT 2 and T 1 AT 2 MP helices (15).
  • Q is in Angstroms
  • phase is in degrees
  • populations are in percent of the total furanose conformations in each of the three DNA strands.
  • Intrastrand phosphate distances of T 1 AT 2 and T 1 AT 2 MP helices The calculated intrastrand phosphate distances (in Angstroms) averaged over the 40 psec trajectory are shown for the entire triple helical systems. Standard interstrand phosphorus distances are 6.0A for A-DNA and 7.0A for B-DNA (15).

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Abstract

Un segment spécifique d'ADN double brin peut être détecté ou reconnu par formation d'une structure à triple hélice en utilisant un oligomère constitué d'unités nucléosides reliées par des liaisons de phosphore internucléoside. La fonction ou l'expression de segments d'ADN double brin peut être empêchée par la formation à triple hélice. De nouveaux oligomères comprenant des unités nucléosides modifiées sont utiles dans la formation à triple hélice et peuvent éventuellement être dérivés avec des groupe de modification de l'ADN.
PCT/US1992/007246 1991-08-30 1992-08-27 Formation de complexes a triple helice d'adn double brin a l'aide d'oligomeres de nucleosides qui comprennent des analogues a base de purine WO1993005180A1 (fr)

Priority Applications (4)

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EP92919326A EP0672171A4 (fr) 1991-08-30 1992-08-27 Formation de complexes a triple helice d'adn double brin a l'aide d'oligomeres de nucleosides qui comprennent des analogues a base de purine.
AU25544/92A AU668886B2 (en) 1991-08-30 1992-08-27 Formation of triple helix complexes of double stranded DNA using nucleoside oligomers which comprise purine base analogs
JP5505283A JPH06510203A (ja) 1991-08-30 1992-08-27 プリン塩基類似体を含むヌクレオシドオリゴマーを用いる二重鎖ｄｎａの三重らせん複合体の形成
FI940922A FI940922L (fi) 1991-08-30 1994-02-25 Kolmoiskierukkakompleksien muodostus kaksijuosteisesta DNA:sta käyttäen nukleosidioligomeereja, jotka sisältävät puriiniemäsanalogeja

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US75181391A 1991-08-30 1991-08-30
US751,813 1991-08-30

Publications (1)

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WO1993005180A1 true WO1993005180A1 (fr) 1993-03-18

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PCT/US1992/007246 WO1993005180A1 (fr) 1991-08-30 1992-08-27 Formation de complexes a triple helice d'adn double brin a l'aide d'oligomeres de nucleosides qui comprennent des analogues a base de purine

Country Status (8)

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EP (1) EP0672171A4 (fr)
JP (1) JPH06510203A (fr)
AU (1) AU668886B2 (fr)
CA (1) CA2116343A1 (fr)
FI (1) FI940922L (fr)
IL (1) IL102983A (fr)
NZ (1) NZ244125A (fr)
WO (1) WO1993005180A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0558634A1 (fr) * 1990-11-23 1993-09-08 Gilead Sciences, Inc. Oligomeres formant un triplex et contenant des bases modifiees
WO1997041140A1 (fr) * 1996-04-26 1997-11-06 Novartis Ag Oligonucleotides modifies
EP1228243A1 (fr) * 1999-10-29 2002-08-07 Cygene, Inc. Compositions, procedes de syntese et utilisation de nouvelles structures d'acides nucleiques
US6441130B1 (en) 1991-05-24 2002-08-27 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
US6451968B1 (en) 1991-05-24 2002-09-17 Isis Pharmaceuticals, Inc. Peptide nucleic acids
US6713602B1 (en) 1991-05-24 2004-03-30 Ole Buchardt Synthetic procedures for peptide nucleic acids
US7148406B2 (en) 1997-08-27 2006-12-12 Pioneer Hi-Bred International, Inc. Genes encoding enzymes for lignin biosynthesis and uses thereof
US7223833B1 (en) 1991-05-24 2007-05-29 Isis Pharmaceuticals, Inc. Peptide nucleic acid conjugates

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0375408A1 (fr) * 1988-12-20 1990-06-27 Baylor College Of Medicine Méthode de préparation d'oligonucléotides synthétiques se liant spécifiquement à des cibles sur des molécules d'ADN bicaténaire en formant un complexe tricaténaire colinéaire, les oligonucléotides synthétiques et méthodes d'utilisation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0478708A4 (en) * 1989-06-19 1993-06-09 The Johns Hopkins University Formation of triple helix complexes of double stranded dna using nucleoside oligomers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0375408A1 (fr) * 1988-12-20 1990-06-27 Baylor College Of Medicine Méthode de préparation d'oligonucléotides synthétiques se liant spécifiquement à des cibles sur des molécules d'ADN bicaténaire en formant un complexe tricaténaire colinéaire, les oligonucléotides synthétiques et méthodes d'utilisation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Journal of the American Chemical Society, Volume 112, issued 1990, D.A. HORNE et al., "Recognition of Mixed Sequence Duplex DNA by Alternative-Strand Triple-Helix Formation", pages 2435-2437, entire document. *
Science, Volume 241, issued 22 July 1988, M. COONEY et al., "Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in Vitro", pages 456-459, entire document. *
Science, Volume 251, issued 15 March 1991, P.A. BEAL et al., "Second Structural Motif for Recognition of DNA by Oligonucleotide-Directed Triple Helix Formation", pages 1360-1363, entire document. *
See also references of EP0672171A4 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0558634A1 (fr) * 1990-11-23 1993-09-08 Gilead Sciences, Inc. Oligomeres formant un triplex et contenant des bases modifiees
EP0558634A4 (en) * 1990-11-23 1995-06-28 Gilead Sciences Inc Triplex-forming oligomers containing modified bases
US6441130B1 (en) 1991-05-24 2002-08-27 Isis Pharmaceuticals, Inc. Linked peptide nucleic acids
US6451968B1 (en) 1991-05-24 2002-09-17 Isis Pharmaceuticals, Inc. Peptide nucleic acids
US6713602B1 (en) 1991-05-24 2004-03-30 Ole Buchardt Synthetic procedures for peptide nucleic acids
US7223833B1 (en) 1991-05-24 2007-05-29 Isis Pharmaceuticals, Inc. Peptide nucleic acid conjugates
WO1997041140A1 (fr) * 1996-04-26 1997-11-06 Novartis Ag Oligonucleotides modifies
US7148406B2 (en) 1997-08-27 2006-12-12 Pioneer Hi-Bred International, Inc. Genes encoding enzymes for lignin biosynthesis and uses thereof
EP1228243A1 (fr) * 1999-10-29 2002-08-07 Cygene, Inc. Compositions, procedes de syntese et utilisation de nouvelles structures d'acides nucleiques
EP1228243A4 (fr) * 1999-10-29 2005-07-20 Cygene Inc Compositions, procedes de syntese et utilisation de nouvelles structures d'acides nucleiques

Also Published As

Publication number Publication date
IL102983A (en) 1998-02-08
IL102983A0 (en) 1993-01-31
AU668886B2 (en) 1996-05-23
CA2116343A1 (fr) 1993-03-18
EP0672171A1 (fr) 1995-09-20
JPH06510203A (ja) 1994-11-17
AU2554492A (en) 1993-04-05
FI940922L (fi) 1994-04-07
NZ244125A (en) 1995-04-27
EP0672171A4 (fr) 1997-04-09
FI940922A0 (fi) 1994-02-25

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