EP0672190A1 - Formation de complexes a trois helices d'acides nucleiques monocatenaires a l'aide d'oligomeres nucleosidiques comprenant des analogues de pyrimidine - Google Patents

Formation de complexes a trois helices d'acides nucleiques monocatenaires a l'aide d'oligomeres nucleosidiques comprenant des analogues de pyrimidine

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EP0672190A1
EP0672190A1 EP94901571A EP94901571A EP0672190A1 EP 0672190 A1 EP0672190 A1 EP 0672190A1 EP 94901571 A EP94901571 A EP 94901571A EP 94901571 A EP94901571 A EP 94901571A EP 0672190 A1 EP0672190 A1 EP 0672190A1
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strand
target sequence
nucleoside
base
sequence
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English (en)
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Paul On-Pong Ts'o
Tina Lynn Trapane
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Johns Hopkins University
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Johns Hopkins University
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-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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    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6839Triple helix formation or other higher order conformations in hybridisation assays
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/10Type of nucleic acid
    • C12N2310/15Nucleic acids forming more than 2 strands, e.g. TFOs
    • C12N2310/152Nucleic acids forming more than 2 strands, e.g. TFOs on a single-stranded target, e.g. fold-back TFOs
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/32Chemical structure of the sugar
    • C12N2310/3212'-O-R Modification
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    • C12N2310/00Structure or type of the nucleic acid
    • C12N2310/30Chemical structure
    • C12N2310/32Chemical structure of the sugar
    • C12N2310/3222'-R Modification

Definitions

  • the present invention is directed to novel methods of detecting, recognizing and/or inhibiting or altering expression of specific sequences in single stranded nucleic acids, particularly RNA, using Second and Third Strands which are capable of specifically complexing with a selected single stranded nucleic acid sequence to give a triple helix complex.
  • DNA triple helical complexes con- taining cytosine and thymidine on the third strand have been reported to be stable in slightly acidic to neutral solutions (pH 5.0-6.5), respectively, but have been reported to dissociate on increasing pH.
  • Incorporation of modified bases of T, such as 5-bromo-uracil, and C, such as 5-methylcytosine, into the third strand has been reported 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, it was thought that a hydrogen must be available on the N-3 of the pyrimidine ring for hydrogen bonding. Accordingly, it has been proposed that cytosine be protonated at N-3.
  • DNA has been reported to exhibit a variety 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.
  • the possibility of developing therapeutic agents which bind to critical regions of the genome and selectively inhibit the function, replication and survival of abnormal cells is an exciting concept. See, e.g.. Dervan, P., Science 232:464-471 (1988) .
  • Various laboratories have pursued the design and development of molecules which interact with DNA in a sequence-specific manner.
  • nuclease-resistant nonionic oligodeoxynucleotides having a methylphosphonate backbone have been studied in vitro and in vivo as potential anticancer, antiviral and antibacterial agents.
  • ODN Nuclease-resistant nonionic oligodeoxynucleotides
  • the 5'-3' linked internucleoside bonds of these analogs are said to approximate the conformation of phosphodiester bonds in nucleic acids.
  • HSV herpes simplex virus
  • Mechanisms of action for inhibition by the MP 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 are reported to be able to selectively inhibit the 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. (See, e.g. , U.S. Patent Nos. 4,469,863 and 4,511,713) .
  • anti-sense olig ⁇ nucleotides or phosphoro- thioate analogs complementary to a part of viral mRNA to interrupt the transcription and translation of viral mRNA into protein.
  • the anti-sense constructs can bind to viral mRNA and were thought to obstruct the cell's ribosomes from moving along the mRNA and thereby halting the translation of mRNA into protein, a process called "translation arrest” or "ribosomal-hybridization arrest.”
  • Yarochan, et al. "AIDS Therapies", Scientific American, pages 110-119 (October, 1988) .
  • the present invention is directed to methods of selectively detecting, recognizing and/or inhibiting or altering expression of a specific target sequence of a single stranded nucleic acid having any selected sequence of nucleosides (e.g., mixed purine and pyrimidine nucleo ⁇ sides) by formation of a triple helix complex wherein a Second Strand specifically binds to the target sequence by Watson Crick base pairing and also specifically hydrogen bonds to a Third Strand.
  • the Second and Third Strands comprise Oligomers that are optionally covalently linked to each other.
  • triple helix complexes may be formed with a target sequence having any selected combination of pyrimidine and purine bases using the hydrogen bonding motifs described herein.
  • nucleosides are used in the Second Strand which have two hydrogen bonding faces, a Watson-Crick binding face and a Second-Third Strand binding face.
  • the Watson- Crick binding face of a Second Strand base hydrogen bonds to the corresponding base of a nucleoside of the target sequence by Watson-Crick base pairing.
  • the Second-Third Stand binding face of a Second Strand base has at least two hydrogen bonding (donor and/or acceptor) sites and specifically hydrogen bonds with and binds to a comple ⁇ mentary base of a corresponding nucleoside of the Third Strand.
  • triplets are formed and formation of multiple triplets gives a triple helix complex.
  • the naturally occurring pyrimidine bases do not have sufficient hydrogen bonding sites on what would be their Second-Third Strand binding faces to stably hydrogen bond with a base of a Third Strand to form a triplet.
  • nucleosides having bases which are analogs of the naturally occurring cytidine and uridine (or thymidine) in the Second Strand in place of those pyrimidines and which, unlike these naturally occurring pyrimidines, have a proton donor or acceptor at the 5-position; both a base of a nucleoside of the target sequence and a base of a nucleoside of the Third Strand can hydrogen bond to the Second Strand base and form a triplet.
  • the base of the corresponding target sequence nucleoside hydrogen bonds with the Watson-Crick binding face of the Second Strand base and the base of the corresponding Third Strand nucleoside hydrogen bonds with the Second-Third Strand binding face of the Second Strand base.
  • pyrimidine analogs advantageously allows formation of triple helix complexes with single stranded nucleic acids having any target sequence (any mixture of pyrimidine and purine nucleosides) without restriction of the target sequence to homopyrimidine or homopurine sequences or sequences having polypyrimidine or polypurine tracts linked together.
  • target sequence any mixture of pyrimidine and purine nucleosides
  • nucleo ⁇ sides having pyrimidine 5-donor/acceptor bases in the Second Strand and a Third Strand selected according to one of motifs I to V of Figure 1 or Figure 2B allows forma ⁇ tion of a triple helix complex with a target sequence which contains any mixture of pyrimidine and purine bases.
  • the present invention is directed to a method of detecting, recognizing or inhibit ⁇ ing or altering expression of a specific target sequence of single stranded nucleic acid having nucleosides comprising both purine and pyrimidine bases.
  • the single stranded nucleic acid is contacted with Second and Third Strands which comprise Oligomers optionally linked together.
  • the Second Strand comprises at least one nucleoside with a pyrimidine 5-donor/acceptor base.
  • the Second Strand is sufficiently complementary to the target sequence and the Third Strand is sufficiently complemen ⁇ tary to the Second Strand to form a triple helix complex by formation of triplets between individual bases of the target sequence and individual bases of each of the Second and Third Strands.
  • the nucleoside sequences of the Second and Third Strands are selected according to one of motifs I to V of Figure 1 or Figures 2A and 2B such that triplets are formed, each triplet comprising a base of a nucleoside of the target sequence hydrogen bonding with a base of a nucleoside of the Second Strand and the Second Strand base hydrogen bonding with both the target strand base and a base of a nucleoside of the Third Strand. Formation of multiple adjacent triplets produces a triple helix complex.
  • the present invention is based upon our innovative finding that by selecting the base sequences of the Second Strand and the Third Strand according to one of the motifs I to V of Figure 1 or Figures 2A and 2B one may specifically detect or recognize a target sequence and form a triple helix complex with the target sequence without regard to its base composition.
  • the Second and Third Strands having base sequences selected in accordance with one of motifs I to V of Figure 1 or Figure 2A and 2B exhibit high specificity and high affinity in recognizing the target sequence and formation of a triple helix complex at physiological pH and temperatures.
  • the present invention provides a Second Strand complementary to the target sequence which binds to the target sequence by Watson-Crick base pairing and which has in place of the naturally occurring pyrimi ⁇ dine bases C and U (or T) , modified pyrimidine bases which have an additional hydrogen bonding site at the 5-position ("pyrimidine-5-donor/acceptor-bases”) .
  • pyrimidine-5-donor/acceptor bases have a structure which allows them to act as a proton donor or acceptor at the 5-* position of the base's ring and which gives the base an additional position for hydrogen -bonding.
  • These pyri ⁇ midine-5-donor/acceptor bases have an additional hydrogen bonding site and therefore can form• hydrogen bonds and, thus, bind to another base (on its Second-Third Strand binding face) .according to one of motifs I to T- of Figure 1 or Figure 2B.
  • These pyrimidine 5-donor/acceptor bases can bind to a base of the target sequence by Watson Crick base pairing on their front side (or “Watson-Crick binding face”) and also form hydrogen bonds on their back side (or "Second-Third Strand binding face") with another base according to one of motifs I to V of Figure 1 or Figure 2B (see "Third Strand Base Selection") to form a triplet.
  • a Second Strand having at least one pyrimidine-5- donor/acceptor base.
  • Suitable pyrimidine-5-donor/acceptor bases include pseudoisocytosine or pseudoisocytosine * (in place of cytosine) and pseudouracil or pseudocytosine (in place of uracil or thymine) .
  • the Second Strand and the Third Strand may be covalently linked and, thus, comprise a single Oligomer.
  • the Second Strand and the Third Strand may each comprise separate Oligomers.
  • the Second and Third Strands comprise substantially neutral Oligomers. Especially preferred substantially neutral Oligomers are methylphosphonate Oligomers.
  • Second and Third Strands each comprise from about 4 to about 40 nucleosides, more preferably from about 6 to about 30 nucleosides. Especially preferred are Second and Third Strands which each comprise about 8 to about 20 nucleosides.
  • the present invention is directed to a Second Strand which is a first Oligomer and which has a nucleoside sequence selected in accordance with one of motifs I to V of Figure 1 or Figure 2A.
  • a Second Strand capable of forming a triple helix complex with a target sequence having a mixture of purine and pyrimidine nucleosides.
  • the Second Strand comprises a plurality of nucleosides wherein the base portion of each nucleoside has a Watson-Crick binding face capable of binding to a base of a nucleoside of the target sequence by Watson-Crick base pairing and a Second- Third Strand binding face having at least two hydrogen binding sites and being capable of binding to a base of a nucleoside of the Third Strand.
  • the present invention is directed to a Third Strand which is a second Oligomer having a nucleoside sequence selected in accordance with one of motifs I to V of Figure 1 or Figure 2B.
  • a base of a nucleoside of each of the Second and Third Strands will interact with each other and the base of the Second Strand will interact with a corres- ponding base of a nucleoside of the target sequence to form a triplet as set forth in one of motifs I to V of Figure 1 or Figure 2A and B. Formation of triplets with bases of multiple adjacent nucleosides of the target sequence result in a triple helix complex.
  • methods are provided of forming a triplet between a purine nucleoside of a target sequence of a single stranded nucleic acid, a corresponding nucleoside of a Second Strand and a corresponding nucleoside of a Third Stand wherein the Second Strand nucleoside comprises a pyrimidine analog which has a Watson-Crick binding face capable of binding by Watson-Crick base pairing to the purine base and a Second-Third Strand binding face having at least two hydrogen binding sites.
  • the purine nucleo ⁇ side of the target sequence is contacted with the Second Strand nucleoside and a Third Strand nucleoside which has a base complementary to the Second-Third Strand binding face of the base of the Second Strand nucleoside to give, a triplet.
  • Preferred target sequences for detection, recognition and/or inhibition or alteration of expression by the Second and Third Strands according to the methods of the present invention have from about 4 to about 40 nucleo ⁇ sides. Sequences of this length are long enough to ' be unique, but are short enough for selectivity towards the target sequence (the Second and Third Strands are unlikely to bind to an unrelated target sequence) .
  • 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, or guanine analogs modified at the 6-position or the analog of adenine, 2-amino purine, as well as analogs of purines having carbon replacing nitrogen at the 9-position such as the 9-deaza purine derivatives and other purine analogs such as those set forth in Figure 4 herein.
  • nucleoside includes a nucleosidyl unit and is used interchangeably therewith, and refers to a subunit of a nucleic acid which comprises a 5-carbon sugar and a nitrogen-containing base.
  • the term includes not only those nucleosidyl units having A, G, C, T and U as their bases, but also analogs and modified forms of the naturally-occurring bases, including the pyrimidine-5- donor/acceptor bases such are pseudoisocytosine and pseudouracil and other modified bases (such as 8- substituted purines) .
  • the 5-carbon sugar is ribose; in DNA, it is a 2' -deoxyribose.
  • nucleoside also includes other analogs of such subunits, including those which have modified sugars such as 2'-0-- alkyl ribose.
  • pyrimidine C-nucleoside In the context of the pseudo ) pyrimidine C-nucleoside, it may be called the “pseudo" (or “ ⁇ ") numbering system, or alternatively just the number of the ring position may be used.
  • the numbering system is shown in figures 3A and 3B wherein each X may be independently nitrogen or carbon.
  • R is hydrogen or an alkyl or aryl group.
  • Suitable alkyl or aryl groups include those which do not sterically hinder the phosphonate linkage or interact with each other.
  • 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.
  • phosphodiester refers to the group
  • phosphodiester groups may be used as internucleosidyl phosphorus group linkages (or links) to connect nucleosidyl units.
  • non-nucleoside monomeric unit refers to a monomeric unit wherein the base, the sugar and/or the phosphorus backbone has been replaced by other chemical moieties.
  • a “nucleoside/non-nucleoside polymer” refers to a polymer comprised of nucleoside and non-nucleoside monomeric units.
  • oligonucleoside or “Oligomer” refers to a chain of nucleosides which are linked by internucleoside linkages which is generally from about 4 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.
  • the term "Oligomer” refers to a chain of oligonucleosides which have internucleosidyl linkages linking the nucleoside monomers and, thus, includes oligonucleotides, nonionic oligonucleoside alkyl- and aryl-phosphonate analogs, alkyl- and aryl- phosphonothioates, phosphorothioate or phosphorodithioate analogs of oligonucleotides, phosphoramidate analogs of oligonucleotides, neutral phosphate ester oligonucleoside analogs, such as phosphotriesters and other oligonucleoside analogs and modified oligonucleosides, and also includes nucleoside/non-nucleoside polymers.
  • nucleoside/nucleotide polymers wherein one or more of the phosphorus group linkages between monomeric units has been replaced by a non-phosphorous linkage such as a formacetal linkage, a sulfamate linkage, or a carbamate linkage. It also includes nucleoside/non- nucleoside polymers wherein both the sugar and the phosphorous moiety have been replaced or modified such as morpholino base analogs, or polyamide base analogs.
  • nucleoside/non-nucleoside polymers wherein the base, the sugar, and the phosphate backbone of the non-nucleoside are either replaced by a non-nucleoside moiety or wherein a non-nucleoside moiety is inserted into the nucleoside/non-nucleoside polymer.
  • said non-nucleoside moiety may serve to link other small molecules which may interact with target sequences or alter uptake into target cells.
  • alkyl- or aryl-phosphonate Oligomer refers to Oligomers having at least one alkyl- or aryl- phosphonate internucleosidyl linkage.
  • Suitable alkyl- or aryl- phosphonate groups include alkyl- or aryl- groups which do not sterically hinder the phosphonate linkage or interact with each other.
  • Preferred alkyl groups include lower alkyl groups having from about 1 to about 6 carbon atoms.
  • Suitable aryl groups have at least one ring having a conjugated pi electron system and include carbocyclic aryl and heterocyclic aryl groups, which may be optionally substituted and preferably having up to about 10 carbon atoms.
  • methylphosphonate Oligomer (or “MP- Oligomer”) refers to Oligomers having at least one methylphosphonate internucleosidyl linkage.
  • neutral Oligomer refers to Oligomers which have nonionic internucleosidyl linkages between nucleoside monomers (i.e., linkages having no positive or negative ionic charge) and include, for example, Oligomers having internucleosidyl linkages such as alkyl- or aryl- phosphonate linkages, alkyl- or aryl-phosphonothioates, neutral phosphate ester linkages ' such as phosphotriester linkages, especially neutral ethyltriester linkages; and non-phosphorus-containing internucleosidyl linkages, such as sulfamate, morpholino, formacetal, and carbamate linkages.
  • internucleosidyl linkages such as alkyl- or aryl- phosphonate linkages, alkyl- or aryl-phosphonothioates
  • neutral phosphate ester linkages ' such as phosphotriester linkages, especially neutral ethy
  • a neutral Oligomer may comprise a conjugate between an oligonucleoside or nucleoside/non- nucleoside polymer and a second molecule which comprises a conjugation partner.
  • conjugation partners may comprise intercalators, alkylating agents, binding substances for cell surface receptors, lipophilic agents, nucleic acid modifying groups including photo-cross- linking agents such as psoralen and groups capable of cleaving a targeted portion of a nucleic acid, and the like.
  • conjugation partners may further enhance the uptake of the Oligomer, modify the interaction of the Oligomer with the target sequence, or alter the pharmacokinetic distribution of the Oligomer.
  • the essential requirement is that the oligonucleoside or nucleoside/non-nucleoside polymer that the Oligomer conjugate comprises be neutral.
  • substantially neutral Oligomer refers to Oligomers in which at least about 80 percent of the internucleosidyl linkages between the nucleoside monomers are nonionic linkages.
  • neutral alkyl- or aryl- phosphonate Oligomer refers to neutral Oligomers having neutral internucleosidyl linkages which comprise at least one alkyl- or aryl- phosphonate linkage.
  • neutral methylphosphonate Oligomer refers to neutral Oligomers having internucleosidyl linkages which comprise at least one methylphosphonate linkage.
  • Second and Third Strands refers to Strands having base sequences which allow the Strand or Oligomer to hydrogen bond with the base sequence of the target sequence of a nucleic acid or another strand and thus bind to the nucleic acid or other strand and in combination to form a triple helix complex.
  • p as listed in ApG represents a phosphodiester internucleo- side linkage and p_ as in Cp_G represents a methylphospho ⁇ nate internucleoside linkage. Also the notation such as T indicates nucleosides linked by methylphosphonate linkages.
  • triplet or "triad” refers a hydrogen bonded complex of three nucleoside bases between a base of a target sequence, a base of a first Oligomer and a base of Oligomer as set forth in one of motifs I to V of
  • Figure 1 depicts triplet formation motifs using the Watson-Crick pairs of permutation 1 of Figure 2A.
  • Figure 2A depicts four possible permutations of Second Strand bases for use in recognizing the naturally occurring bases of the target sequence. These Second Strand bases have at least two hydrogen bonding positions on their Second-Third Strand binding faces so as to be able to selectively hydrogen bond with a base of a Third Strand nucleoside.
  • Figure 2B depicts motifs I to V for selection of Third Strand bases dependent on the hydrogen bonding pattern of the Second-Third Strand binding face of the Second Strand base.
  • Figure 3A depicts the numbering system used herein for pyrimidine and pyrimidine analog bases .
  • Figure 3B depicts the ring numbering system used herein for purine and purine analog bases.
  • Figure 4 depicts the structures of and abbreviation for certain bases used according to the methods of the present invention to form triple helix complexes.
  • Figure 5 depicts the Watson-Crick base paring schemes for permutation 1 of Figures 2A.
  • Figure 6 depicts the Watson-Crick base pairing schemes for permutation 4 of Figure 2A.
  • Figure 7 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif I .
  • Figure 8 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif I' .
  • Figure 9 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif II.
  • Figure 10 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif II' .
  • Figure 11 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif III.
  • Figure 12 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif III' .
  • Figure 12 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif IV.
  • Figure 14 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif IV .
  • Figure 15 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif V.
  • Figure 16 depicts triads formed with the Watson-Crick base pairs of permutation 1 according to motif V .
  • the antisense strategy for the development of speci- fically synthesized oligonucleotides (and their analogs) as sequence-specific/gene-specific therapeutic agents has now become a major direction for drug development.
  • Two conflicting/contradictory demands in the design of the Second and Third Strands are of primary concern.
  • the therapeutic Second and Third Strands should be absolutely sequence-specific for their designated target sequences in order to avoid unwanted interactions with the large number of other nucleic acid sequences within the cells. Adven ⁇ titious interactions of administered Oligomers with other sequences could lead to undesirable side effects.
  • there is a requirement of high affinity of the Oligomers for the target so that low concentrations for the Oligomers will be sufficient for the masking function at the target site.
  • the on-rate should be controlled by diffusion and will be influenced by the accessibility of the target sequence of nucleic acid to the Oligomers and only slightly by the size (number of monomeric units) of the Oligomers .
  • the kinetic element in living processes is a very vital concern. All of the reactions and interactions in living cells are related to each other functionally in a kinetic manner, and are not necessarily related to each other in a thermodynamic manner. If an inappropriate binding exists too long because of an insufficient off-rate, then damage to the cell may have sufficient time to occur, thus leading to undesirable biological effects. Therefore, on one hand, we need to have a rapid process of search and determination during the initial phase of interaction, but, on the other hand, we need a very slow process of dissociation once the correct binding between the probe and the target nucleic acid occurs.
  • the first step is "Search".
  • the major objective, in this step is for the Oligomers to rapidly screen interactions with all of the possible nucleic acid targets inside the living cells and tissues as quickly as possible, with a relatively fast off-rate.
  • a second step is now required for the "Sealing", leading to the formation of a complex which has a very slow off- rate.
  • the psoralen on the Oligomer can form a cyclobutane-type of crosslinking with a double bond in a pyrimidine base, for example, cytosine or uracil located in the target strand only in a perfectly matched duplex. Since the Oligomer will be covalently linked to the target nucleic acid in the perfectly matched duplex upon photo-irradiation, the off-rate is now practically reduced to zero for the covalent complex. The challenge is to be able to form a similar type of complex in two steps, but to eliminate the requirement for an external energy source, such as photo-irradiation.
  • pseudo-isocytosine nucleoside With these alternative bases, an appropriate hydrogen-bonding site is provided in the neutral unproto- nated base for triple helix formation.
  • pseudo- 25 isocytosine in the third strand to form triple helix complexes is described in our co-pending application, United States Serial No. 07/772,081.
  • each Oligomer participates in sequence specific hydrogen-bonding with the target strand and the Oligomers (Second and Third Strands) do not participate in hydrogen- bonding with each other; i.e. the target sequence is enclosed by hydrogen-bonding interactions with the Oli ⁇ gomers.
  • This type of arrangement is termed a closed triplex or "closed sandwich” because the target sequence is sandwiched between two Oligomers.
  • the "open sandwich” arrangement using naturally occurring bases can be formed when the target sequence consists only of pyrimidine residues.
  • sequence specific Watson-Crick inter- actions are satisfied by a homopurine Oligomer (Second Strand) .
  • this Second Strand must interact with a Third Strand at its C6, N7 face.
  • the Second Strand makes sequence specific hydrogen-bonds with the target sequence and the Second and Third Strands share a hydrogen-bonding interface and hydrogen bond with each other.
  • the target strand is on an open side of the triple helix complex. This second type of arrangement is termed an open triple helix or "open sandwich" .
  • a closed sandwich may be a more favorable arrangement than an open sandwich.
  • This understanding is somewhat intuitive as the target strand must break away from two sets of hydrogen-bonding interactions with the Oligomers in the closed sandwich case, whereas there is only one such set of interactions to break in the open sandwich case.
  • this consi- deration may not be relevant when the target nucleic acid is a large molecule with only a small segment of the target sequence in a single-stranded form, and, thereby, available for sequence-specific complex formation.
  • the open sandwich arrangement may be preferred.
  • the Second Strand is bound as the Watson-Crick complement to the target sequence and is restricted from dissociating by the added Third Strand.
  • This Third Strand binding may decrease the dissociation constant of the complex by 100 to 1000 fold, and could reduce the needed concentration for therapeutic action,* for instance, from 100 ⁇ M to 1.0 or 0.1 ⁇ M. More importantly, the length requirement for the available open sequence of the target nucleic acid can still be relatively short, such as from 10 to 14 nucleotide units. The target nucleic acid is much more likely to have an open single-stranded region of such a length, instead of a longer ( ⁇ 20 nucleotides) sequence.
  • triple helix complexes may be formed with any sequence arrangement of the single-stranded nucleic acid target.
  • the major limitation in the triple helix formation has been that the pyrimidine in the Watson-Crick duplex has only one additional hydrogen bonding site after the formation of the duplex via Watson-Crick hydrogen bonding scheme.
  • One aspect of the present invention is to use C- nucleosides for the pyrimidines in the Second Strand of the Watson-Crick duplex formed with the target sequence in replacement of the naturally occurring N-nucleoside.
  • the glycosidic bond between the pyrimidine base and the sugar moiety is a carbon-carbon bond
  • the N-nucleosides the pyrimidine base and sugar are attached by a nitrogen-carbon bond.
  • the C-pyrimidine nucleoside has an additional hydrogen bonding site for a pair of hydrogen bond formation with the third strand added to the Watson-Crick duplex. Since there is only a small change in the C-C bond vs. the C-N bond distance (about 0.IA) , the original nucleic acid structure is preserved with minimal perturbation. In this manner, the usefulness of C- pyrimidine nucleosides is greatly magnified for a triple helix formation as compared to the naturally occurring N- pyrimidine nucleoside.
  • the present invention provides a compre ⁇ hensive approach for triple helix formation with target nucleic acid sequences consisting of any combination of the four naturally occurring bases which is described below.
  • the bases of some of the nucleosides proposed for use in the Second and Third Strands are naturally occurr- ing minor bases, such as pseudo-uracil, and xanthine and are commercially available; syntheses for other of the unusual bases have appeared in the literature; and yet other may be prepared by syntheses analogous to literature syntheses. (See included references given in "Nucleoside Bases" herein below) .
  • Figures 7 to 16 depict triads formed according to motifs I to V of Figure 1 (or Permutation 1 of Figure 2A) .
  • Second Strand incorporates in place of the naturally occurring cytidine or uridine (or thymidine) nucleosides, analogs of these nucleosides which are able to form Watson-Crick base pairs with the target sequence, but also have an additional hydrogen bonding site at the position which corresponds to the 5-position of cytidine or uridine, which we have termed pyrimidine-5-donor/ acceptor bases nucleosides.
  • Preferred pyrimidine-5-donor/acceptor bases nucleo ⁇ sides include C-nucleosides, that is where the glycosidic bond is attached to a carbon atom of the heterocyclic base, rather than to a ring nitrogen. Attachment to a carbon atom allows the ring nitrogen to be available for hydrogen bonding.
  • the proposed hydrogen bonding patterns and isomorphic geometries requires the use of several nonstandard (i.e. not naturally occurring) heterocyclic bases.
  • the atom numbering for the naturally occurring purine and pyrimidine nucleosides is set forth in Figures 3A and 3B.
  • the atom numbering follows that for the standard bases.
  • the covalent attachment to the sugar is at the N9 position for purines and at Nl for pyrimidines.
  • the target strand base will be indicated first in bold with the complementary base in the Second Strand separated by a bullet (•) representing Watson-Crick hydrogen bonding schemes in each pair.
  • Recognition of target sequence pyrimidine bases by Second Strand bases gives the standard base pairs C-»G and U*A.
  • recognition of target sequence purine bases is accomplished by C- nucleoside pyrimidine bases on the Second Strand to give the base pairs, G « iC and A « U.
  • the hydrogen bonding pattern for these "pseudo Watson-Crick" base pairs is the same as for the standard G ⁇ »C and A*U base pairs.
  • OG has two acceptors
  • G* iC has two donors
  • U*A has a donor and an acceptor (as viewed from the major groove)
  • A*" ⁇ U has an acceptor and a donor.
  • nucleosides (or bases) for the Third Strand may be based on one of triad motifs I to V of Figures 1 and 2B. These motifs are based upon Third Strand recognition by either pyrimidine or purine nucleosides and are separated into three classes according to their general recognition schemes. Systematic con ⁇ struction and ordering of these motifs will be according to the following set of guidelines. First, it is assumed that all nucleosides on the Third Strand will have the anti configuration of the base at the glycosidic linkage. Therefore, proposed Third Strand polarities can be made directly by comparison to the Watson-Crick strands. Second, a pair of specific hydrogen-bonds must be made to the Watson-Crick Second Strand by adjacent donor/acceptor sites on the Third Strand base.
  • pyrimidine bases possess two sets of adjacent sites (C4-N3 and N3-C2) whereas purine bases have three (C6-N1, N1-C2 and C6-N7) .
  • the overall form of the base triads should be geometrically isomorphous. Because the target strand may contain a heterologous sequence of bases, the Watson-Crick section of the base triads will have the familiar pseudo dyad symmetry of the base pair.
  • the third strand may be only of one type of base (i.e., all pyrimidine or all purine) . This requirement is important to the formation of triple- stranded helices in order to ensure regular positioning of the Third Strand backbone and to optimize stacking interactions between adjacent triads.
  • Motifs I, I', II and II' (Class A motifs) are constructed using pyrimidine Third Strand bases .
  • the Third Strand T (at 04 and H3) accepts and donates a hydrogen-bond to A (at H6 and N7) .
  • This type of hydrogen-bonding scheme was first identified by Hoogsteen in crystals of 1-methylthymine and 9-methyladenine. The strand polarities of triple helices containing only this triad has been shown to be anti- parallel for the Watson-Crick interaction and parallel for the Hoogsteen interaction.
  • the isomorphic triads in this motif are generated by identifying pyrimidine bases which possess the requisite hydrogen-bond donor/acceptor pairs at C4 and N3 or at comparable position in ⁇ -pyrimidines .
  • Recognition of the Second Strand at G requires two donors on the Third Strand. This is provided by the iC base (note that the tautomeric form is now different than that required for Watson-Crick recognition by this base) or alternatively the iC * base.
  • the following discussion is directed to permutation 1 of Figure 2A which is more fully depicted in Figure 1.
  • the individual triads of motifs I to V for permutation 1 are depicted in Figures 7 to 16.
  • Recognition of Second Strand iC requires two acceptors provided by the natural base analog iC.
  • the triad completing motif I involves recognition of U by C which provides a donor and acceptor (again, as viewed from the major groove of the Watson-Crick helix) .
  • Motif I' is related to motif I by an inversion of the polarity of the Third Strand backbone, maintaining Third Strand recognition by donor-acceptor sites at C4 and N3 positions of the Third Strand pyrimidine bases.
  • base pairing involving two donors to two acceptors can be constructed by flipping the orientation of the base (thereby the orientation of the backbone) for motif I 180° in the plane of the paper so that the donor/acceptor at N3 (or " ⁇ N3) is now hydrogen-bonding at the site on the Second Strand base closest to the major groove and the substituent at C4 (or C4) is now hydrogen- bonding toward the minor groove.
  • the proposed base triad motifs include a subset which can be utilized to recognize naturally occurring double-stranded target pyrimidine* purine sequences.
  • the base pairs C «G and T*A found in DNA are equivalent to the third and fourth Watson-Crick pairs in each motif (See Figure 1) . Therefore, it may be possible to form triple-stranded helices at double- stranded target sites by the addition of a single oligomer probe designed according to the rules of the ten motifs presented here. For example, it was described in United States Serial No.
  • base substitutions may be made in any motif as long as the specific hydrogen bonding patterns are maintained and the new triad remains isomorphic to the remaining triads in the motif.
  • Third Strand recognition by purine bases in motifs III, III', IV and IV does not involve hydrogen-bonding at N7. (See, e.g. , Figures 11 to 14) . Therefore, it may be advantageous to synthesize some or all of the Third Strand residues as 7- deaza- analogs (derivatives of tubercidin) in order to avoid unwanted interactions at this face of the Third Strand residues as 7- deaza- analogs (derivatives of tubercidin) in order to avoid unwanted interactions at this face of the Third Strand residues as 7- deaza- analogs (derivatives of tubercidin) in order to avoid unwanted interactions at this face of the Third Strand residues as 7- deaza- analogs (derivatives of tubercidin) in order to avoid unwanted interactions at this face of the Third
  • sugar moiety and backbone linkages of the Oligomer probe strands can be any that are available.
  • the choice of these elements for the backbone should be made based upon their ability to confer chemical stability and favorable characteristics in terms of binding stability and specificity. Obviously, a number of choices are available regarding both sugar and backbone linkage.
  • Common sugar moieties include 2' -deoxyribose, ribose, or 2' -O-methylribose.
  • Suitable backbones for the Third Strand include phosphodiester, methylphosphonate or phosphorothioate.
  • the hydrogen bonding patterns for the Second-Third Strand binding face are depicted by the double arrow or pair of arrows to the right of the Second Strand base.
  • the triads for motifs I to V using Second Strand bases selected according to permutation 1 of Figure 2A and Third Strand bases selected according to motifs I to V are depicted in Figures 7 to 16.
  • Figure 2B depicts the Third Strand binding motifs for each of the four specified Second-Third Strand binding patterns. Strand polarity of target, Second and Third strand are indicated in the right hand column.
  • Second and Third Strands Base sequences for appropriate Second and Third Strands to form a triple helix complex with a target strand having any combination of pyrimidine nucleosides may be conveniently determined using Figures 2A and 2B. Second and Third Strands
  • the Second and Third Strands may comprise separate Oligomers. Alternatively, the Second and Third Strands may be covalently linked together. Preferably the Second and Third Strands each comprise from about 4 to about 40 nucleosides, more preferably from about 6 to about 30 nucleosides and especially preferred are Second and Third Strands of about 8 to about 20 nucleosides.
  • the Second and Third Strands of the present invention comprise optionally covalently linked Oligomers.
  • Oligomers having the desired internucleoside linkages may be conveniently prepared according to synthetic techniques known to those skilled in the art. For example, commercial machines, reagents and protocols are available for the synthesis of Oligomers having phosphodiester and certain other phosphorus-containing internucleoside linkages. See also Gait, M.J., Oligonucleotide Synthesis: A Practical Approach (IRL)
  • the Second and Third Strands of the present invention may include certain analogues of the naturally occurring pyrimidine and purine bases. These analogs include the above-noted pyrimidine-5- donor/acceptor bases. The synthesis of these bases used in our proposed binding motifs have been reported and by following those literature procedures, those bases can be made.
  • Pseudouridine ( ⁇ U) is commercially available (from Kyowa Hakko Kogyo Co. Ltd., N.Y.) .
  • the synthesis of pseudocytidine is reported by Pankiewicz, K.W. , et al. , Carbohyd. Res. 127:227-233 (1984) .
  • Isoguanosine * (IG * ) is synthesized by methods analogous to those reported by Revanker et al . , J. Med. Chem. 22:1389 (1984) for 3-deazaguanine.
  • Inosine (I) and its phosphoramidite synthon are commercially available (from Cruachem, Herndon, VA) .
  • Inosine * (I * ) and isoinosine * (ii * ) may be prepared by methods analogous to those reported by Rosemeyer and Seela, J. Org. Chem. 52:5136-5143 (1987) for 5-aza-7- deazaguanosine.
  • Xanthine (X) and Xanthosine are commercially available (from Sigma) .
  • the synthesis of 2-amino-purine ( 2a P) is reported by McLaughlin, L.W. , et al . , Nucl . Acids Res. 16 :5631-5644 (1988) and by Doudna, J.A. , et al. , J. Org. Chem. 55 :5547- 5549 (1990) .
  • Second and Third Strands that each have a corresponding nucleoside complementary to each nucleoside of the target sequence (i.e., have "exact complementarity") .
  • Second and Third Strands which may lack a complement for each nucleoside in the target sequence, provided that the Second Strand has such binding affinity for the target sequence and the Third Strand has sufficient binding affinity for the Second Strand that together the Second and Third Strands bind with the target sequence to recognize it or to inhibit its expression by forming a triple helix complex.
  • Such strands are referred to as being “substantially complementary” or having "substantial complementarity”.
  • the Second Strand should be substantially comple ⁇ mentary to the target sequence and the Third Strand should be substantially complementary to the Second Strand in that there is sufficient hybridization and hydrogen- bonding between the strands for inhibition of expression of the target sequence, and if the target sequence is a portion of a mRNA, inhibition of translation, to occur. Sufficient hybridization and hydrogen-bonding is related to the strength of the hydrogen-bonding between bases as well as the specificity of the complementary strand.
  • the strength of the hydrogen-bonding is influenced by the number and percentage of bases in a strand that are base paired to complementary bases, according to either Watson- Crick base pairing (for target-Second Strand binding) or between Second Strand and Third Strand, whether by previously described triplet formation schemes or by one of triad motifs I to V .
  • the complemen- tary bases of the strand must be sufficient in number so as to avoid non-specific binding to other sequences within a genome and while at the same time small enough in number to avoid non-specific binding between other sequences within a genome and portions of a long strand.
  • the base sequence of either the Second or the Third Strand need not be 100 percent complementary to the sequence to which it is to bind. Preferably the sequence is at least about 80 percent complementary, more preferably at least about 90 percent and even more preferably about 95 percent or more.
  • the Second and Third Strand may optionally include one or more non-nucleoside monomeric units. Such non-nucleoside monomeric units include those described in co-pending U.S. Serial No. 07/565,307, filed August 9, 1990 (also pub ⁇ lished PCT Application No. WO 92/02532) , the disclosure of which is incorporated herein by reference.
  • the strand in question need only be capable of sufficient hybridization or bonding to the target sequence (or the Second Strand) to prevent or interfere with expression of the target sequence, such as by preventing normal translation of the target sequence or to specifically recognize the target sequence.
  • Prevention of normal translation of the target sequence occurs when an expression product of the target sequence is produced in an amount significantly lower than would be the result in the absence of the Second and Third Strands.
  • the expression product is a protein. Measure ⁇ ment of the decrease in production of proteins is well known to those skilled in the art and such methods include quantification by chromatography, biological assay or immunological reactivity.
  • a specific seg ⁇ ment of single stranded nucleic acid may be detected or recognized using Second and Third Strands which form a triple helix with the single stranded nucleic acid according to the triplet base pairing guidelines described herein.
  • the Second and Third Strands have sequences selected as described above such that a base of the Second Strand will hydrogen bond with a base of the target sequence (by its Watson-Crick binding face) and with a corresponding base of the Third Strand (by its Second- Third Strand binding face) to give a triplet and, thus, to result in a triple helix complex.
  • the Second and Third Strands are Oligomers which may be optionally covalently linked. Detectably labeled Oligomers may be used as proved for use in hybridization assays, for example, to detect the presence of a particular single-stranded nucleic acid sequence.
  • the present invention also provides a method of preventing or altering expression or function of a selected target sequence of single stranded nucleic acid by use of Second and Third Strands which form a triple stranded helix structure with the single stranded target as described above. Formation of the triple stranded helix may prevent expression and/or function by modes such as preventing transcription, preventing of binding of effector molecules (such as proteins) , etc.
  • a high affinity complex is formed with a high degree of selectivity.
  • Derivatized Second and Third Strands may be used to detect or locate and then irreversibly modify the target site in the nucleic acid by cross-linking (psoralens) or cleaving one or both strands (EDTA) .
  • psoralens cross-linking
  • EDTA cleaving one or both strands
  • one of the strands may be used as a molecular scissors to specifically excise a selected nucleic acid sequence.
  • the Second or Third Strands may be derivatized to incorporate a nucleic acid reacting or modifying group which can be caused to react with the nucleic acid segment or a target sequence thereof to irreversibly modify, degrade or destroy the nucleic acid and thus irreversibly inhibit its functions.
  • These Second and Third Strands may be used to inactivate or inhibit or alter expression of a particular gene or target sequence of the same in a living cell, allowing selective inactivation or inhibition or alteration of expression.
  • the target sequence may be DNA or RNA, such as a pre-mRNA, an mRNA or an RNA sequence such as an initiator codon, a polyadenylation region, an mRNA cap site or a splice junction. These strands 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 single stranded nucleic acid sequence which comprises Second and Third Strands at least one of which is detectably labeled and selected to be able sufficiently complementary to the target sequence of the single stranded nucleic acid to be able to form a triple helix structure therewith.
  • Antisense therapy is a generic term which includes the use of specific binding Oligomers to inactivate undesirable DNA or RNA sequences in vitro or in vivo.
  • Antisense therapy includes targeting a specific DNA or RNA target sequence through complementarity or through any other specific binding means, in the case of the present invention by formation of triple helix complexes according to the binding motifs described herein.
  • the Oligomers for use in the instant invention may be administered singly, or combinations of Oligomers may be administered for adjacent or distant targets or for combined effects of antisense mechanisms with the foregoing general mechanisms.
  • the Oligomers can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, .latest edition.
  • the Oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which may include fillers, extenders, binding, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of administration and dosage forms.
  • Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
  • injection may be preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the Oligomers for use with the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the Oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds, can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucusal administration.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through use of nasal sprays, for example, as well as formulations suitable for administration by inhalation, or suppositories.
  • the Oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the Oligomers for use in the invention are formulated into ointments, salves, eye drops, gels, or creams, as is generally known in the art.
  • the methods of the present invention may be used diagnostically to detect the presence or absence of the target DNA or RNA sequences to which the Oligomers specifically bind. Such diagnostic tests are conducted by hybridization through triple helix complex formation which is then detected by conventional means.
  • Oligomers may be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support detected.
  • the presence of a triple helix may be detected by antibodies which specifically recognize forms. Means for conducting assays using such Oligomers as probes are generally known.

Abstract

Cette invention concerne des procédés de détection, de reconnaissance et/ou d'inhibition ou de modification de l'expression d'une séquence cible d'un acide nucléique monocaténaire comprenant n'importe quelle combinaison de nucléosides de purine et de pyrimidine, selon lesquels on forme des complexes à trois hélices en combinaison avec des deuxième et troisième brins présentant des oligomères facultativement liés de manière covalente.
EP94901571A 1992-11-18 1993-11-17 Formation de complexes a trois helices d'acides nucleiques monocatenaires a l'aide d'oligomeres nucleosidiques comprenant des analogues de pyrimidine Withdrawn EP0672190A1 (fr)

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