WO2011102414A1 - Dérivé oligonucléotidique - Google Patents

Dérivé oligonucléotidique Download PDF

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WO2011102414A1
WO2011102414A1 PCT/JP2011/053373 JP2011053373W WO2011102414A1 WO 2011102414 A1 WO2011102414 A1 WO 2011102414A1 JP 2011053373 W JP2011053373 W JP 2011053373W WO 2011102414 A1 WO2011102414 A1 WO 2011102414A1
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general formula
group
carbon atoms
represented
oligonucleotide derivative
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PCT/JP2011/053373
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English (en)
Japanese (ja)
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康志 清尾
光雄 関根
一也 宮崎
早耶子 黒萩
恵理佳 兒玉
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国立大学法人 東京工業大学
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Priority to JP2012500643A priority Critical patent/JPWO2011102414A1/ja
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    • 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
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N15/111—General methods applicable to biologically active non-coding 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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

Definitions

  • the present invention relates to an oligonucleotide derivative, and particularly relates to an oligonucleotide having two or more substituents capable of dissociating into anions at the 5 ′ end or 3 ′ end of the molecule.
  • the oligonucleotide derivative of the present invention shows a strong binding force to RNA having a sequence complementary to itself, but shows weak binding to RNA having a sequence complementary to itself at the non-terminal portion. . That is, it has a short RNA binding ability, which shows a strong binding force to short RNAs but only weak bonds to long RNAs.
  • These properties are useful as a probe nucleic acid in a technique for selectively detecting an active substance such as microRNA, which acts as an active RNA after being cut short from a long precursor RNA in a cell.
  • MicroRNA is RNA transcribed from the genome of an organism and usually has a chain length of about 19 to 23 bases, and in some cases about 15 to 30 bases, which does not encode a protein. Recent research suggests that miRNA plays an important role in processes such as cell development, differentiation and canceration, and viral infection processes. The mechanism of production of active mature miRNA is as follows.
  • a pri-miRNA of about 1000 bases transcribed from the genome is cleaved to generate a pre-miRNA of 70-100 bases.
  • This pre-miRNA is further cleaved to produce an active mature miRNA of about 19 to 23 bases.
  • This mature miRNA binds to the target messenger RNA and controls its translation, thereby regulating the expression level of the protein synthesized based on the information of the messenger RNA.
  • Non-patent Document 1 A method for detecting only RNA is known (Non-patent Document 1). According to this method, since a long-chain nucleic acid is not contained in a sample, only a short-chain nucleic acid can be analyzed. However, it takes time and cost to perform column chromatography and electrophoresis, and operation is difficult. There is a problem that it is difficult to implement unless it is a skilled engineer due to its complexity.
  • Non-patent document 2 a method of using a fluorescent label of a nucleic acid using an enzymatic reaction capable of chain extension only when short-chain RNA is used as a primer in order to omit the molecular process of short-chain RNA has been reported ( Non-patent document 2).
  • this method uses a plurality of enzyme reactions, there is a problem that the operation becomes complicated.
  • Non-Patent Document 3 discloses a mature miRNA selective RT-PCR method using a hairpin primer that binds only to a short RNA.
  • hairpin primers need to be precisely designed for thermodynamic stability of the hairpin structure, and are not necessarily universal and easy to use.
  • RT-PCR is not an optimal method in terms of throughput because it is difficult to comprehensively detect many miRNAs present in cells.
  • the root cause of the above problem is that natural and non-natural oligonucleotides that have existed so far can selectively recognize the sequence of a nucleic acid molecule, but cannot recognize its size. That is, since an oligonucleotide complementary to a certain sequence also binds to a long nucleic acid molecule that includes the target sequence as a part thereof, an RNA that includes the mature miRNA sequence as a part thereof, such as pre-miRNA, It is difficult to distinguish mature RNA from hybridization.
  • RNA that includes the mature miRNA sequence as a part thereof such as pre-miRNA
  • Non-patent Document 4 a mature RNA selective microarray in which a hairpin type probe is immobilized on a microarray has been reported.
  • the document describes that only mature miRNAs that have been processed short by this hairpin probe can be recognized, but in order to form a hairpin structure in the probe, the length of the probe must be made longer than necessary. Therefore, it is not an optimal method when considering the cost of probe synthesis. Also, the quantitative evaluation of how much the hairpin primer actually binds to the mature miRNA is not sufficient.
  • a method for detecting only short RNA by introducing a sterically bulky substituent at both ends of the oligonucleotide probe has been reported (Non-patent Document 5). It is not a low and practical method.
  • Patent Document 1 also reports a method of detecting only short RNAs by introducing sterically bulky substituents at both ends of the oligonucleotide probe, but has low selectivity for binding to short RNAs. Not practical.
  • Patent Document 2 discloses an oligonucleotide derivative in which a substituent having a phosphate group is introduced into the terminal nucleobase moiety. If this oligonucleotide derivative is used, only short-chain RNA can be detected. However, an oligonucleotide derivative with higher selectivity for short RNA is desired.
  • An object of the present invention is to provide an oligonucleotide derivative that binds only to a short complementary strand and does not bind to a long complementary strand RNA or complementary DNA.
  • the present inventors have solved the above problems by using an oligonucleotide derivative having two or more substituents capable of dissociating into anions at the end of the oligonucleotide. I got the knowledge that I get.
  • the present invention has been made based on the above findings, and provides an oligonucleotide derivative represented by the general formula (1) and a salt thereof.
  • R 1 is a hydroxyl group or a group represented by the following general formula (2)
  • W 1 is a natural or non-natural nucleobase or the following general formula (3)
  • general formula (5) Y 1 represents a hydroxyl group, a hydrogen atom, a fluorine atom or an alkoxy group which may have a substituent
  • Y 2 represents a group represented by any one of General Formula (6) and General Formula (7)
  • X k is the same or different for each k value
  • Each represents a hydroxyl group, a hydrogen atom, a fluorine atom or an alkoxy group which may have a substituent
  • B k may be the same or different for each value of k, Represents a natural or non-natural nucleobase
  • k is an integer of 1 to 50 Represents
  • W 2 is
  • R 2 is a hydroxyl group or a group represented by the general formula (2) (provided that at least one of R 1 , R 2 or Y 2 is a group represented by the general formula (2);
  • W 1 is any one of general formula (3), general formula (5), general formula (6), or general formula (7);
  • General formula (5) is any of formulas (6) or formula (7))
  • Q 3 and T 3 may be the same or different and each represents an oxygen atom or a sulfur atom
  • R 3 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
  • L 1 represents an alkane-diyl group having 2 to 6 carbon atoms, a cycloalkane-diyl group having 4 to 8 carbon atoms
  • R 4 represents
  • Q 4 and T 4 may be the same or different and each represents an oxygen atom or a sulfur atom
  • R 4 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
  • Z represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
  • L 2 represents an alkane having 2 to 6 carbon atoms.
  • R 5 represents a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a general formula (It represents a group represented by (4) or an alkyl group having 1 to 6 carbon atoms having a group represented by the general formula (4) at its terminal.)
  • R 6 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms
  • L 3 represents an alkane-diyl group having 2 to 6 carbon atoms, a cycloalkane-diyl group having 4 to 8 carbon atoms
  • R 7 represents a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl
  • L 4 and L 5 may be the same or different from each other, and may be an alkane-diyl group having 2 to 6 carbon atoms, a cycloalkane-diyl group having 4 to 8 carbon atoms, a phenylene group, or Represents a naphthylidene group
  • R 8 and R 9 may be the same or different from each other, a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a group represented by the general formula (4) Or an alkyl group having 1 to 6 carbon atoms having a group represented by the general formula (4) at the terminal.
  • R 1 is a group represented by the general formula (2), Q 3 and T 3 in the general formula (2) are oxygen atoms, and W 1 is the general formula (3).
  • R 2 and / or Y 2 is a group represented by the general formula (2), Q 3 and T 3 in the general formula (2) are oxygen atoms
  • W 2 is A group represented by general formula (3), general formula (5), general formula (6) or general formula (7), wherein Q 4 and T 4 in general formula (4) are oxygen atoms.
  • R 1 is a phosphate group
  • W 1 is a group represented by general formula (3), general formula (5), general formula (6) or general formula (7).
  • Q 4 and T 4 are oxygen atoms.
  • R 2 and / or Y 2 is a phosphate group
  • W 2 is represented by general formula (3), general formula (5), general formula (6) or general formula (7).
  • R 2 and / or Y 2 is a phosphate group
  • W 2 is the general formula (3), general formula (5), general formula (6), or general formula (7).
  • Q 4 and T 4 in the general formula (4) are oxygen atoms.
  • the oligonucleotide derivative of the present invention is preferably used as a probe for nucleic acid detection.
  • the present invention also provides an oligonucleotide array in which the oligonucleotide derivative is immobilized on a solid support.
  • the present invention also provides a pharmaceutical composition comprising the above oligonucleotide derivative.
  • the present invention also provides reverse transcriptase primers, DNA polymerase primers, and DNA polymerase templates useful for PCR and RT-PCR.
  • the oligonucleotide derivative of the present invention Since the oligonucleotide derivative of the present invention has two or more substituents capable of dissociating into anions at the terminal portion thereof, by setting appropriate reaction conditions, it binds only to a short complementary strand, It is an oligonucleotide derivative that does not bind to long complementary RNA or DNA.
  • the oligonucleotide derivative of the present invention by reacting the oligonucleotide derivative of the present invention with nucleic acids having various chain lengths and sequences extracted from cells, it binds only to nucleic acids having a complementary sequence to the added oligonucleotide and having a short chain length. And can be used for isolation, detection, and function control of a nucleic acid having a short chain length.
  • the oligonucleotide derivative of the present invention is represented by the following general formula (1).
  • R 1 is a hydroxyl group or a group represented by the following general formula (2).
  • Q 3 and T 3 may be the same or different and each represents an oxygen atom or a sulfur atom.
  • Q 3 and T 3 are oxygen atoms. That is, one in which R 1 in the general formula (1) is a phosphoric acid group can be mentioned.
  • W 1 is a natural or non-natural nucleobase or any one of the following general formula (3), general formula (5), general formula (6), or general formula (7). Represents the group to be represented.
  • Examples of natural nucleobases include thymin-1-yl, cytosine-1-yl, 5-methyl-cytosyn-1-yl, uracil-1-yl, uracil-5-yl, adenine-9-yl, Examples include guanine-9-yl.
  • the non-natural nucleobase is a chemical modification such as having a substituent on the exocyclic oxygen atom, exocyclic nitrogen atom, cyclin nitrogen atom or carbocyclic carbon atom of the natural nucleobase, or exocyclic oxygen.
  • R 3 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, butan-2-yl, n-pentyl, and n-hexyl.
  • L 1 represents an alkane-diyl group having 2 to 6 carbon atoms, a cycloalkane-diyl group having 4 to 8 carbon atoms, a phenylene group, or a naphthylidene group.
  • the alkane-diyl group represents an alkanediyl group having 2 to 6 carbon atoms, for example, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane- 1,6-diyl and the like can be mentioned. Also included are branched groups such as butane-1,3-diyl.
  • the cycloalkane-diyl group means a divalent group derived from a cycloalkane having 4 to 8 carbon atoms, for example, cyclobutane-1,3-diyl, cyclobutane-1,2-diyl, cyclopentane-1 , 3-diyl, cyclopentane-1,2-diyl, cyclohexane-1,2-diyl, cyclohexane-1,3-diyl, cyclohexane-1,4-diyl, etc.
  • groups both are included.
  • the phenylene group means a divalent group derived from benzene, and includes 1,2-phenylene, 1,3-phenylene, 1,4-phenylene and the like.
  • the naphthylidene group means a divalent group derived from naphthalene.
  • 1,2-naphthylidene, 1,3-naphthylidene, 1,4-naphthylidene, 1,5-naphthylidene, 1,6-naphthylidene, 1,7- Examples include naphthylidene, 1,8-naphthylidene, 2,3-naphthylidene, 2,6-naphthylidene, 2,7-naphthylidene, and the like.
  • R 4 is a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a group represented by the general formula (4), or a group having 1 to carbon atoms having a group represented by the general formula (4) at the terminal.
  • an alkyl group is mentioned as an alkyl group.
  • Q 4 and T 4 may be the same as or different from each other, and represent an oxygen atom or a sulfur atom.
  • R 4 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, butan-2-yl, n-pentyl, and n-hexyl.
  • Z represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • alkyl group examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, butan-2-yl, n-pentyl, and n-hexyl.
  • L 2 represents an alkane-diyl group having 2 to 6 carbon atoms, a cycloalkane-diyl group having 4 to 8 carbon atoms, a phenylene group, or a naphthylidene group.
  • the alkane-diyl group represents an alkanediyl group having 2 to 6 carbon atoms, for example, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane- 1,6-diyl and the like can be mentioned. Also included are branched groups such as butane-1,3-diyl.
  • the cycloalkane-diyl group means a divalent group derived from a cycloalkane having 4 to 8 carbon atoms, for example, cyclobutane-1,3-diyl, cyclobutane-1,2-diyl, cyclopentane-1 , 3-diyl, cyclopentane-1,2-diyl, cyclohexane-1,2-diyl, cyclohexane-1,3-diyl, cyclohexane-1,4-diyl, etc.
  • groups both are included.
  • the phenylene group means a divalent group derived from benzene, and includes 1,2-phenylene, 1,3-phenylene, 1,4-phenylene and the like.
  • the naphthylidene group means a divalent group derived from naphthalene.
  • 1,2-naphthylidene, 1,3-naphthylidene, 1,4-naphthylidene, 1,5-naphthylidene, 1,6-naphthylidene, 1,7- Examples include naphthylidene, 1,8-naphthylidene, 2,3-naphthylidene, 2,6-naphthylidene, 2,7-naphthylidene, and the like.
  • R 5 is a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a group represented by the general formula (4), or a group having 1 to carbon atoms having a group represented by the general formula (4) at the terminal.
  • R 6 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms.
  • alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, butan-2-yl, n-pentyl, and n-hexyl.
  • L 3 represents an alkane-diyl group having 2 to 6 carbon atoms, a cycloalkane-diyl group having 4 to 8 carbon atoms, a phenylene group, or a naphthylidene group.
  • the alkane-diyl group represents an alkanediyl group having 2 to 6 carbon atoms, for example, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane- 1,6-diyl and the like can be mentioned. Also included are branched groups such as butane-1,3-diyl.
  • the cycloalkane-diyl group means a divalent group derived from a cycloalkane having 4 to 8 carbon atoms, for example, cyclobutane-1,3-diyl, cyclobutane-1,2-diyl, cyclopentane-1 , 3-diyl, cyclopentane-1,2-diyl, cyclohexane-1,2-diyl, cyclohexane-1,3-diyl, cyclohexane-1,4-diyl, etc.
  • groups both are included.
  • the phenylene group means a divalent group derived from benzene, and includes 1,2-phenylene, 1,3-phenylene, 1,4-phenylene and the like.
  • the naphthylidene group means a divalent group derived from naphthalene.
  • 1,2-naphthylidene, 1,3-naphthylidene, 1,4-naphthylidene, 1,5-naphthylidene, 1,6-naphthylidene, 1,7- Examples include naphthylidene, 1,8-naphthylidene, 2,3-naphthylidene, 2,6-naphthylidene, 2,7-naphthylidene, and the like.
  • R 7 is a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a group represented by the general formula (4), or a group having 1 to carbon atoms having a group represented by the general formula (4) at the terminal.
  • L 4 and L 5 may be the same or different, and the alkane-diyl group having 2 to 6 carbon atoms, the cycloalkane-diyl group having 4 to 8 carbon atoms, phenylene Group or a naphthylidene group.
  • the alkane-diyl group represents an alkanediyl group having 2 to 6 carbon atoms, for example, ethylene, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, hexane- 1,6-diyl and the like can be mentioned. Also included are branched groups such as butane-1,3-diyl.
  • the cycloalkane-diyl group means a divalent group derived from a cycloalkane having 4 to 8 carbon atoms, for example, cyclobutane-1,3-diyl, cyclobutane-1,2-diyl, cyclopentane-1 , 3-diyl, cyclopentane-1,2-diyl, cyclohexane-1,2-diyl, cyclohexane-1,3-diyl, cyclohexane-1,4-diyl, etc.
  • groups both are included.
  • the phenylene group means a divalent group derived from benzene, and includes 1,2-phenylene, 1,3-phenylene, 1,4-phenylene and the like.
  • the naphthylidene group means a divalent group derived from naphthalene.
  • 1,2-naphthylidene, 1,3-naphthylidene, 1,4-naphthylidene, 1,5-naphthylidene, 1,6-naphthylidene, 1,7- Examples include naphthylidene, 1,8-naphthylidene, 2,3-naphthylidene, 2,6-naphthylidene, 2,7-naphthylidene, and the like.
  • R 8 and R 9 may be the same or different, and may be a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a group represented by the general formula (3), or a general terminal. This represents an alkyl group having 1 to 6 carbon atoms having a group represented by the formula (3).
  • an alkyl group may be the same or different, and may be a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a group represented by the general formula (3), or a general terminal. This represents an alkyl group having 1 to 6 carbon atoms having a group represented by the formula (3).
  • an alkyl group may be a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, a group represented by the general formula (3), or a general terminal.
  • Y 1 represents a hydroxyl group, a hydrogen atom, a fluorine atom, or an alkoxy group that may have a substituent.
  • the alkoxy group is preferably an alkoxy group having 1 to 6 carbon atoms. Examples of such an alkoxy group include a methoxy group, an ethoxy group, a propoxy group, a propan-1-yloxy group, and a butan-1-yloxy group. 1-butyloxy group, 1-pentyloxy group, 1-hexyloxy group and the like, and branched alkoxy groups such as 2-propyloxy group, isobutyloxy group, cyclopropyloxy group, cyclobutyl, etc.
  • alkoxy group in which a part or all of the side chain is cyclized such as an oxy group, a cyclopentyloxy group, a cyclohexyloxy group, and a cyclopropylmethyloxy group is also included.
  • substituents include bromo, chloro, iodo, hydroxyl, cyano, alkoxy, amino, alkylamino, dialkylamino, aryl, heteroaryl and the like.
  • Y 2 is a hydroxyl group, a hydrogen atom, a fluorine atom, an alkoxy group which may have a substituent, or a group represented by the general formula (2).
  • alkoxy group include those described above.
  • General formula (2) is as described above.
  • substituent include bromo, chloro, iodo, hydroxyl, cyano, alkoxy, amino, alkylamino, dialkylamino, aryl, heteroaryl and the like.
  • X k may be the same as or different from each value of k, and each alkoxy group may have a hydroxyl group, a hydrogen atom, a fluorine atom, or a substituent.
  • the alkoxy group include those described above.
  • the substituent include bromo, chloro, iodo, hydroxyl, cyano, alkoxy, amino, alkylamino, dialkylamino, aryl, heteroaryl and the like.
  • B k may be the same or different for each value of k, and each represents a natural or non-natural nucleobase.
  • the natural or unnatural nucleobase include those described in W 1.
  • B k in the general formula (1) such as a sequence complementary to the nucleic acid to be detected can be chosen such that the sequence with the appropriate cohesive strength.
  • k is an integer of 1 to 50, preferably 5 to 30.
  • k can be changed depending on the chain length of the RNA (miRNA or the like) to be detected. That is, the oligonucleotide derivative of the present invention has two or more substituents that can be dissociated into anions at the terminal portion, and the complementary strand RNA or complementary strand DNA is obtained by dissociating these substituents into anions. Electrostatic repulsion occurs and long chain lengths cannot be bound, and only DNA strands and RNA strands that are shorter than the length of the anion at the end are bound to the oligonucleotide derivative of the present invention. Will be able to. Therefore, it is possible to vary the range of k depending on the length of the miRNA or the like to be detected.
  • W 2 is a natural or non-natural nucleobase, or any one of the general formula (3), the general formula (5), the general formula (6), or the general formula (7). Represents the group to be represented. Natural or unnatural nucleobase, general formula (3), for the general formula (5), the general formula (6), or the general formula (7) are the same as those described in W 1.
  • Q 1 and Q 2 may be the same or different and each represents an oxygen atom or a sulfur atom.
  • T 1 and T 2 may be the same or different and each represents an oxygen atom or a sulfur atom.
  • R 2 is a hydroxyl group or a group represented by the general formula (2).
  • General formula (2) is as described above.
  • R 1 , R 2 or Y 2 is a group represented by the general formula (2).
  • W 1 represents the general formula (3), the general formula (5), the general formula (6), or the general formula ( 7);
  • R 2 or Y 2 is a group represented by the general formula (2), W 2 represents the general formula (3), the general formula (5), or the general formula (6).
  • a group represented by general formula (7) That is, the oligonucleotide derivative of the present invention has two or more substituents capable of dissociating into anions at the 5 ′ end or 3 ′ end of the molecule.
  • R 1 is a group represented by the general formula (2)
  • W 1 is represented by any one of the general formula (3), the general formula (5), the general formula (6), or the general formula (7). It is a group. That is, when R 1 is a group represented by the general formula (2), W 1 represents a carboxyl group, an alkyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal, represented by the general formula (4). Or an alkyl group having 1 to 6 carbon atoms having a group represented by the general formula (4) at the terminal.
  • the oligonucleotide derivative molecule has two phosphate groups at its 5 ′ end, or has a phosphate group and a carboxylic acid group, so it can dissociate into an anion at the 5 ′ end. Two substituents are included.
  • W 2 is any one of the general formula (3), the general formula (5), the general formula (6), or the general formula (7).
  • W 1 represents a carboxyl group having 1 to 6 carbon atoms having a carboxyl group at the terminal.
  • Two or more substituents capable of dissociating into anions are included.
  • both R 2 and Y 2 are groups represented by the general formula (2)
  • the 3 ′ end of the oligonucleotide derivative molecule contains three substituents that can dissociate into anions; Become.
  • Q 3 and T 3 in the general formula (2) and Q 4 and T 4 in the general formula (4) are oxygen atoms, that is, represented by the general formula (2).
  • a group in which the group is a phosphate group.
  • both R 1 and R 2 may be groups (or phosphate groups) represented by the general formula (2).
  • the oligonucleotide derivative of the present invention may be in the form of its salt.
  • the salt used in the present specification means a compound in which protons of some or all of the phosphate groups and carboxyl groups contained in the nucleotide derivatives of the present invention are replaced with cations.
  • Examples of the cation include ammonium ions such as ammonium, monoalkylammonium, dialkylammonium, trialkylammonium, and tetraalkylammonium, and sodium ions, potassium ions, lithium ions, calcium ions, magnesium ions, manganese ions, and iron ions. And metal ions such as copper ions.
  • the oligonucleotide derivative of the present invention has two or more substituents capable of dissociating into anions at its 5 ′ end, 3 ′ end, or both ends, so that appropriate reaction conditions are set. By doing so, an anion is generated at the terminal portion.
  • a method for detecting only a short complementary strand using the oligonucleotide derivative of the present invention will be described with reference to the drawings.
  • FIG. 1 is a diagram schematically showing a method for detecting only a short complementary strand using the oligonucleotide derivative of the present invention.
  • the oligonucleotide derivative of the present invention generates an anion at the end under appropriate reaction conditions.
  • a short complementary strand here, the short complementary strand means a complementary strand having a sequence complementary to the oligonucleotide derivative of the present invention at the end,
  • the complementary strand means a complementary strand having a sequence complementary to the oligonucleotide derivative of the present invention at a non-terminal portion.
  • the long complementary strand even though it has a complementary portion that binds to the oligonucleotide derivative of the present invention, there are two or more anions at the end of the oligonucleotide derivative of the present invention. Electrostatic repulsion occurs at this portion, and the long complementary strand cannot bind to the oligonucleotide derivative of the present invention.
  • the oligonucleotide derivative of the present invention reacts with nucleic acids having various chain lengths and sequences extracted from cells, thereby having a sequence complementary to the oligonucleotide of the present invention.
  • the oligonucleotide derivative of the present invention can be used as a nucleic acid detection probe.
  • suitable reaction conditions are well-known in the said technical field, and mean the conditions for hybridizing a probe and oligonucleotide.
  • Hybridization may be performed in a 96-well or 384-well plastic plate. The oligonucleotide of the present invention is spotted in the hole of such a plate, and then a sample is added to perform hybridization. Or you may use the oligonucleotide array of this invention mentioned later. Hybridization is preferably performed in the temperature range of room temperature to about 70 ° C. for 6 to 20 hours.
  • oligonucleotide derivative of the present invention can be produced by a method known to those skilled in the art, for example, a phosphoramidite method. When carrying out the phosphoramidite method, the oligonucleotide derivative of the present invention can be produced by using a known phosphoramidite compound. Specifically, it can be synthesized according to the method described in the examples of this specification.
  • the oligonucleotide derivative of the present invention can be used as a primer for a reverse transcriptase reaction.
  • it can be applied to the RT-PCR method.
  • RT-PCR method For example, by performing reverse transcription on a sample containing RNA in the presence of the oligonucleotide derivative of the present invention, only miRNA is reverse transcribed and cDNA is synthesized. The obtained cDNA is synthesized by RT-PCR. Thus, DNA having a portion complementary only to miRNA can be obtained.
  • Conventionally known methods can be used for reverse transcription, RT-PCR conditions and the like.
  • the oligonucleotide derivative of the present invention can be further used for oligonucleotide arrays, pharmaceuticals and the like as described below. Furthermore, the oligonucleotide derivative of the present invention can also be used as a primer for reverse transcriptase, a primer for DNA polymerase, and a template for DNA polymerase useful for PCR and RT-PCR.
  • the oligonucleotide array of the present invention is formed by immobilizing the oligonucleotide derivative of the present invention on a solid support.
  • the oligonucleotide array of the present invention is used to detect an oligonucleotide having a specific base sequence and a short chain length (usually 50 bases or less). Therefore, the base sequence of the oligonucleotide derivative immobilized on the solid phase carrier is a sequence having an appropriate binding force, such as one having a sequence complementary to the sequence to be detected.
  • an oligonucleotide derivative immobilized on a solid phase carrier binds to an oligonucleotide to be detected by specific binding through hybridization.
  • the oligonucleotide array can be produced by synthesizing the oligonucleotide directly on the solid support surface (on-chip method) or by immobilizing the prepared oligonucleotide on the solid support surface.
  • the method is known.
  • the oligonucleotide array in the present invention can be produced by any of these methods.
  • an on-chip method for example, a combination of the use of a protective group that is selectively removed by light irradiation, and a photolithographic technique and a solid-phase synthesis technique that are used in semiconductor manufacturing can be used in a predetermined region of a minute matrix. (Masking technology: for example, Fodor, S.P.A.
  • Oligonucleotides are generally covalently bound to a surface-treated solid support via a spacer or a crosslinker.
  • a method is also known in which polyacrylamide gel tiny pieces are aligned on a glass surface, and a synthetic oligonucleotide is covalently bound thereto (Yershov, G. et al. Proc. Natl. Acad. Sci. USA 94: 4913, 1996).
  • an oligonucleotide is synthesized in advance on a solid phase carrier such as porous glass or polystyrene by a solid phase synthesis method, and the solid phase carrier is immobilized on a glass substrate by an appropriate method such as an adhesive or a physical method.
  • an appropriate method such as an adhesive or a physical method.
  • the solid phase carrier to be used, those conventionally used for producing DNA chips and gene detection microarrays can be used without particular limitation.
  • the solid support used include: silicon; glass such as microporous glass and porous glass; metal; magnetic beads having ferrite as a core and the surface covered with glycine methacrylate; plastic (for example, polyester resin, polyethylene resin, polypropylene) Resin, acrylonitrile butadiene styrene resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin) and the like.
  • the shape of the carrier may be any shape such as a plate shape (substrate shape), a bead shape, a thread shape, a spherical shape, a polygonal shape, and a powder shape.
  • the solid phase carrier may be one in which a surface treatment layer of diamond-like carbon is formed.
  • Diamond-like carbon (DLC, Diamond ⁇ Like Carbon) is a general term for an incomplete diamond structure that is a mixture of diamond and carbon, and the mixing ratio is not particularly limited.
  • the thickness of the layer is preferably 1 nm to 10 ⁇ m.
  • oligonucleotide of the present invention in order to covalently bond the oligonucleotide of the present invention to the solid phase carrier, those having an amino group bound to the surface of the solid phase carrier may be used. Therefore, it is preferable to use a carrier having an amino group bonded to the surface or capable of bonding an amino group.
  • the pharmaceutical composition of the present invention contains the above-described oligonucleotide derivative of the present invention.
  • the oligonucleotide derivative of the present invention binds only to a short-chain complementary strand and can be used to control the function of such a short-chain complementary strand.
  • the present invention provides a pharmaceutical composition comprising the above-described oligonucleotide derivative of the present invention.
  • the pharmaceutical composition of the present invention can be produced by a known method such as mixing a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention itself or mixed with appropriate pharmacologically acceptable excipients, diluents, etc., and as tablets, capsules, granules, powders, syrups, etc., or injections , Suppositories, patches, or external preparations.
  • the pharmaceutical composition includes excipients (eg, sugar derivatives such as lactose, sucrose, sucrose, mannitol, sorbitol; starches such as corn starch, potato starch, ⁇ starch, dextrin; crystalline cellulose; gum arabic; dextran; pullulan; Silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium metasilicate aluminate; calcium hydrogen phosphate; calcium carbonate; calcium sulfate, lubricant (eg stearic acid, calcium stearate, Magnesium stearate, talc, colloidal silica, sodium lauryl sulfate, magnesium lauryl sulfate), binders (eg, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, macrogol , Disintegrants (eg low substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, internally cross
  • a colloidal dispersion system For the method of introducing the pharmaceutical composition of the present invention into a subject, a colloidal dispersion system may be used.
  • the colloidal dispersion system has an effect of enhancing the stability of the compound in the living body and an effect of efficiently transporting the compound to a specific organ, tissue or cell.
  • the colloidal dispersion system is not particularly limited as long as it is usually used, and is based on lipids including polymer composites, nanocapsules, microspheres, beads, and oil-in-water emulsifiers, micelles, mixed micelles and liposomes. And a dispersion system.
  • unilamellar liposomes having a size of 0.2 to 0.4 ⁇ m can encapsulate a considerable proportion of an aqueous buffer containing macromolecules, and the oligonucleotide derivative is encapsulated in this aqueous inner membrane.
  • the composition of the liposome is usually a complex of a lipid, particularly a phospholipid (preferably a phospholipid having a high phase transition temperature), with a steroid, particularly cholesterol.
  • lipids useful for the production of liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipid, phosphatidylethanolamine, cerebroside and ganglioside.
  • phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipid, phosphatidylethanolamine, cerebroside and ganglioside.
  • the dose of the pharmaceutical composition containing the oligonucleotide derivative of the present invention varies depending on symptoms and age, but in the case of oral administration, the lower limit is 1 mg (preferably 30 mg) and the upper limit is 2000 mg (preferably, In the case of injection, a lower limit of 0.1 mg (preferably 5 mg) and an upper limit of 1000 mg (preferably 500 mg) can be administered by subcutaneous injection, intramuscular injection or intravenous injection.
  • Raw material synthesis example 1 Synthesis of 3′-5′-O-bis- (tert-butyldimethylsilyl) -6-N- [N- (trans-4-hydroxycyclohexyl) carbamoyl] -2′-O-methyladenosine 3′-5 ′ -O-bis- (tert-butyldimethylsilyl) -2'-O-methyladenosine (1.6 g, 3.1 mmol) was dissolved in 40 mL of pyridine, and then phenyl chloroformate (0.85 mL, 6.8).
  • Raw material synthesis example 2 3′-5′-O-bis- (tert-butyldimethylsilyl) -6-N- [N- [trans-4- (4-oxopentanoyloxy) cyclohexyl] carbamoyl] -2′-O-methyladenosine
  • the compound (2.1 g, 3.2 mmol) obtained in the raw material synthesis example 1 was dissolved in 30 mL of dichloromethane, and levulinic acid (0.72 mL, 6.3 mmol), N, N′-dicyclohexylcarbodiimide (1 .3 g, 6.3 mmol) and 4-dimethylaminopyridine (40 mg, 0.32 mmol) were added, and the mixture was stirred at room temperature for 21 hours.
  • Raw material synthesis example 3 6-N- [N- (trans-4- (4-oxopentanoyloxy) cyclohexyl] carbamoyl] -2′-O-methyladenosine
  • the compound obtained in Raw Material Synthesis Example 2 (1.6 g, 2.1 mmol) ) was dissolved in 20 mL of pyridine, triethylamine trihydrofluoride (1.8 mL, 11 mmol) and triethylamine (1.5 mL, 11 mmol) were added thereto, and the mixture was stirred at room temperature for 3 days.
  • the reaction system was chloroform (50 mL).
  • Raw material synthesis example 4 5'-O- (4,4'-dimethoxytrityl) -6-N- [N- (trans-4- (4-oxopentanoyloxy) cyclohexyl) carbamoyl] -2'-O-methyladenosine
  • Raw material synthesis example 3 The compound obtained in step (0.98 g, 1.9 mmol) was dissolved in 10 mL of pyridine, and 4,4′-dimethoxytrityl chloride (0.77 g, 2.3 mmol) was added thereto, followed by stirring at room temperature for 3 hours. did.
  • Raw material synthesis example 5 5′-O- (4,4′-dimethoxytrityl) -6-N- [N- (trans-4- (4-oxopentanoyloxy) cyclohexyl) carbamoyl] -2′-O-methyladenosine 3 ′-( 2-cyanoethyl N, N-diisopropyl phosphoramidite)
  • the compound obtained in Raw Material Synthesis Example 4 (250 mg, 0.30 mmol) was dissolved in 4.4 mL of dichloromethane, and diisopropylethylamine (261 ⁇ L, 1.5 mmol), 2-cyanoethyldiisopropylchlorophosphoramidite (177 mg) was dissolved therein.
  • Example 1 Synthesis of oligonucleotide 5 ′-(dA *) CAACCUACU- 3 ′ (SEQ ID NO: 1)
  • the underlined nucleotide residue represents a 2′-O-methyl-ribonucleotide residue
  • dA * is It represents a nucleotide residue represented by the following formula.
  • the solid phase carrier was transferred to a glass filter, and the DMTr group was removed by washing the solid phase carrier with 3% trichloroacetic acid diluted with methylene chloride.
  • phosphalink reagent 50 equivalents purchased from Glen Research was reacted for 2 minutes in the presence of 1H-tetrazole (100 equivalents). The reaction with the phosphalink reagent was repeated once more, followed by iodine oxidation by a conventional method.
  • the solid phase carrier was treated with 3% trichloroacetic acid diluted with methylene chloride to remove the DMTr group, and then concentrated aqueous ammonia. And stirred at room temperature for 17 hours. After the aqueous ammonia was distilled off, the resulting crude product was purified by anion exchange HPLC to give the title compound.
  • the structure of the title compound was confirmed by MALDI-TOF mass spectrometry. 5 ′-(dA *) CAACCUACU- 3 ′ (SEQ ID NO: 1) MALDI-TOF mass Calcd 3498.63 Found 3500.03
  • Example 2 Synthesis of Oligonucleotide 5 ′-(dA *) CAACCTACT- 3 ′ (SEQ ID NO: 2)
  • the underlined nucleotide residue represents a deoxyribonucleotide residue.
  • the title compound was synthesized in the same manner as in Example 1 except that DNA phosphoramidite was used instead of 2′-O-methyl RNA phosphoramidite.
  • the structure of the target product was confirmed by MALDI-TOF mass spectrometry.
  • 5 ′-(dA *) CAACCTACT- 3 ′ (SEQ ID NO: 2) MALDI-TOF mass Calcd 32566.5711 Found 3258.81
  • a solid phase carrier (1.0 ⁇ mol) supporting 2′-O-methyluridine purchased from Glen Research and 2′-O-methyl RNA phosphoramidite reagent purchased from Glen Research, ABI-392DNA / Synthesis of 5'- CAACCCUACU having a DMTr group at the 5'-terminal hydroxyl group and a protected nucleobase and phosphate moiety on a solid support using a standard 1.0 ⁇ mol scale RNA synthesis protocol of an RNA synthesizer did.
  • half of the solid support (0.5 ⁇ mol) was transferred to a glass filter, and the DMTr group was removed by washing the solid support with 3% trichloroacetic acid diluted with methylene chloride.
  • phosphalink reagent 50 equivalents purchased from Glen Research was reacted for 2 minutes in the presence of 1H-tetrazole (100 equivalents). The reaction with the phosphalink reagent was repeated once more, followed by iodine oxidation by a conventional method.
  • the solid support was treated with 3% trichloroacetic acid diluted with methylene chloride to remove the DMTr group, and then concentrated aqueous ammonia was added. And stirred at room temperature for 17 hours. After the aqueous ammonia was distilled off, the obtained crude product was purified by anion exchange HPLC to obtain the desired product.
  • the structure of the target product was confirmed by MALDI-TOF mass spectrometry. 5 '-(Am *) CAACCUACU- 3' (SEQ ID NO: 3) MALDI-TOF mass Calcd 35288.6455 Found 3530.316
  • trans-4-aminocyclohexanol (3.6 g, 31.3 mmol) was added without performing the extraction operation, and the mixture was reacted at 85 ° C. for 30 minutes.
  • the solvent was distilled off under reduced pressure and dissolved in chloroform, followed by extraction with saturated sodium bicarbonate.
  • the organic layer was collected, dried by adding sodium sulfate, filtered, and the solvent was distilled off under reduced pressure.
  • the resulting residue was purified by NH silica gel chromatography using hexane / chloroform as a developing solvent to obtain the title compound (2.95 g, 76%) represented by the following formula.
  • Phosphoramidite compound 5 (5′-O- (4,4′-dimethoxytrityl) -N6- [N- (trans-4- (4-oxopentanoyloxy) cyclohexyl) carbamoyl] deoxyadenosine 3 ′-(2 Synthesis of —Cyanoethyl N, N-diisopropylphosphoramidite) Compound 4 (350 mg, 0.441 mmol) was azeotroped three times each in the order of pyridine (dehydration), toluene (dehydration), and dichloromethane (dehydration) Dissolved in 4.4 ml of dichloromethane (dehydrated), tetrazole (19 mg, 0.264 mmol), diisopropylamine (37 ⁇ l, 0.264 mmol) and 2-cyanoethyl-bis (N, N-diisopropyl)- Add phosphoramidite (154 ⁇ l, 0.4
  • Example 4 Analysis of chain length-dependent hybridization by Tm measurement
  • the oligonucleotides obtained in Example 1, Example 2, Example 3 and Comparative Example 1 were added in 10 mM phosphate buffer (pH 7.0) and 0.1 M NaCl. The concentration was adjusted to 1.0 ⁇ M.
  • the following long-chain RNA (SEQ ID NO: 5) or short-chain RNA (SEQ ID NO: 6) is similarly dissolved to a concentration of 1.0 ⁇ M, and the temperature dependency of absorbance at 260 nm is changed from 5 ° C. to 90 ° C. Measured to ° C.
  • the obtained UV melting curve was differentiated, and the temperature at which the first derivative became a maximum was defined as Tm.
  • Tm of the following natural type probe and non-anionic probe was also measured. The results are shown in Table 1.
  • Examples 5 to 10 The same operation as in Example 2 was performed to synthesize the following oligonucleotides. 5 ′-(dA *) TAACCTACT-3 ′ (SEQ ID NO: 9, Example 5): MALDI-TOF mass Calcd 3271.57 Found 3277.29 5 ′-(dA *) CTACCTACT-3 ′ (SEQ ID NO: 10, Example 6): MALDI-TOF mass Calcd 3247.56 Found 3247.56 5 ′-(dA *) CGACTACTACT-3 ′ (SEQ ID NO: 11, Example 7): MALDI-TOF mass Calcd 3272.57 Found 3274.48 5 ′-(dA *) CCACCCTACT-3 ′ (SEQ ID NO: 12, Example 8): MALDI-TOF mass Calcd 3232.56 Found 3234.88 5 ′-(dA *) TTACCTACT-3 ′ (SEQ ID NO: 13, Example 9): MALDI-TOF mass Calcd 32
  • Example 11 Analysis of chain length-dependent hybridization by Tm measurement
  • concentration of the oligonucleotide obtained in Examples 5 to 11 is 1.0 ⁇ M in 10 mM phosphate buffer (pH 7.0) and 0.1 M NaCl. It was prepared as follows. In this solution, the following long RNAs or short RNAs were similarly dissolved to a concentration of 1.0 ⁇ M, and the temperature dependence of absorbance at 260 nm was measured from 5 ° C. to 90 ° C. The obtained UV melting curve was differentiated, and the temperature at which the first derivative became a maximum was defined as Tm.
  • the long RNAs or short RNAs used are shown in Table 2, and the results are shown in Table 3.

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Abstract

L'invention concerne un dérivé oligonucléotidique qui s'associe uniquement avec un brin complémentaire court et qui ne s'associe pas à l'ARN de brin complémentaire long ou à l'ADN complémentaire. Le dérivé oligonucléotidique est représenté par la formule générale (1). Le dérivé oligonucléotidique fait preuve d'une force d'association élevée avec l'ARN court, mais fait preuve uniquement d'une force d'association faible avec l'ARN long. Ces propriétés sont utiles pour servir de sonde d'acide nucléique dans un procédé de détection d'ARN de manière sélective qui fonctionne comme ARN actif après le clivage de l'ARN précurseur long dans une cellule en petit ARN, par exemple, une substance active telle qu'un microARN. L'invention porte, en outre, sur le dérivé oligonucléotidique qui est utilisé comme sonde de détection d'acide nucléique et est également utilisé dans un ensemble oligonucléotidique et dans une composition pharmaceutique.
PCT/JP2011/053373 2010-02-19 2011-02-17 Dérivé oligonucléotidique WO2011102414A1 (fr)

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JP2007238550A (ja) * 2006-03-10 2007-09-20 Tokyo Institute Of Technology オリゴヌクレオチド誘導体
JP2009190983A (ja) * 2008-02-12 2009-08-27 Tokyo Institute Of Technology オリゴヌクレオチド誘導体

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007238550A (ja) * 2006-03-10 2007-09-20 Tokyo Institute Of Technology オリゴヌクレオチド誘導体
JP2009190983A (ja) * 2008-02-12 2009-08-27 Tokyo Institute Of Technology オリゴヌクレオチド誘導体

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