WO2010021344A1 - Nouveau dérivé d'acide nucléique et procédé de préparation d'un acide nucléique résistant aux nucléases par son utilisation - Google Patents

Nouveau dérivé d'acide nucléique et procédé de préparation d'un acide nucléique résistant aux nucléases par son utilisation Download PDF

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WO2010021344A1
WO2010021344A1 PCT/JP2009/064522 JP2009064522W WO2010021344A1 WO 2010021344 A1 WO2010021344 A1 WO 2010021344A1 JP 2009064522 W JP2009064522 W JP 2009064522W WO 2010021344 A1 WO2010021344 A1 WO 2010021344A1
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正靖 ▲桑▼原
聡 小比賀
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独立行政法人科学技術振興機構
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12

Definitions

  • the present invention relates to a novel nucleic acid derivative and a method for preparing a nuclease resistant nucleic acid using the same. More specifically, the present invention relates to a method for imparting nuclease resistance to an existing functional nucleic acid later.
  • nucleic acid molecules are used in various fields such as molecular biology and chemical biology, and their application to pharmaceuticals and diagnostics is being actively studied.
  • nuclease nucleolytic enzyme
  • resistance must be considered. Therefore, even for a nucleic acid molecule for which a clinical trial to sufficiently perform its function in vitro has been obtained, it is necessary to redesign the molecule and impart resistance to the nucleic acid molecule.
  • nuclease resistance is improved by introducing cross-linked nucleotides into functional nucleic acids (antisense, aptamer, etc.).
  • a locked nucleic acid LNA
  • an oligonucleotide having this LNA has improved resistance to nuclease compared to a natural oligonucleotide, and therefore It has been suggested that the stability in the body may be improved (Non-Patent Document 1).
  • Non-Patent Documents 2 to 4 various cross-linked nucleic acids (Bridged Nucleic Acid) that can confer nuclease resistance have been designed and synthesized.
  • the primer containing a triphosphate analog of such a bridging nucleotide has been studied for its influence on DNA polymerase, and it has been reported that modification of the sugar moiety has a large effect on the polymerase reaction (non-patented). Reference 5).
  • LNA which is a bridged nucleotide
  • TdT terminal deoxynucleotide transferase
  • An object of the present invention is to provide a method for easily imparting excellent nuclease resistance to an existing functional nucleic acid later, and to provide a novel nucleic acid derivative for use therein.
  • TdT terminal deoxynucleotide transferase
  • DNA deoxyribonucleotide
  • the present invention provides a method for preparing a nuclease resistant nucleic acid, the method comprising: Mixing and incubating a nucleic acid comprising at least three bases, a terminal deoxynucleotide transferase, and a 2 ′, 4′-bridged nucleoside triphosphate, and
  • the 2 ′, 4′-bridged nucleoside triphosphate is represented by the following formula I, formula II or formula III:
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more arbitrary substituents selected from the following ⁇ group
  • the ⁇ group includes a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and a carbon number.
  • R is selected from a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms that may form a branch or a ring, and the ⁇ group.
  • An aryl group having 3 to 12 carbon atoms which may contain a hetero atom, or a carbon which may contain a hetero atom which may have one or more arbitrary substituents selected from the ⁇ group Represents an aralkyl group having an aryl moiety of 3 to 12;
  • R 1 and R 2 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, and a C 2 to 7 group that may form a branch or a ring.
  • the Base is a 6-aminopurin-9-yl group, a 2,6-diaminopurin-9-yl group, a 2-amino-6-chloro group.
  • R is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group.
  • n 1
  • the Base is a 2,4-dihydroxy-5-methylpyrimidin-1-yl group.
  • either one of R 1 and R 2 is a hydrogen atom.
  • either one of R 1 and R 2 is a phenyl group.
  • the present invention also provides a kit for the preparation of a nuclease resistant nucleic acid comprising a terminal deoxynucleotide transferase and a 2 ′, 4′-bridged nucleoside triphosphate,
  • the 2 ′, 4′-bridged nucleoside triphosphate is represented by the following formula I, formula II or formula III:
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more arbitrary substituents selected from the following ⁇ group
  • the ⁇ group includes a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and a carbon number.
  • R is selected from a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms that may form a branch or a ring, and the ⁇ group.
  • An aryl group having 3 to 12 carbon atoms which may contain a hetero atom, or a carbon which may contain a hetero atom which may have one or more arbitrary substituents selected from the ⁇ group Represents an aralkyl group having an aryl moiety of 3 to 12;
  • R 1 and R 2 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, and a C 2 to 7 group that may form a branch or a ring.
  • the present invention also provides a 2 ', 4'-bridged nucleoside triphosphate represented by the following formula III:
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more arbitrary substituents selected from the following ⁇ group
  • the ⁇ group includes a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and a carbon number.
  • R 1 and R 2 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, and a C 2 to 7 group that may form a branch or a ring.
  • R 1 and R 2 are not simultaneously hydrogen atoms;
  • N is an integer from 1 to 5).
  • the present invention also provides a 2 ', 4'-bridged nucleotide represented by the following formula I', formula II 'or formula III':
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more arbitrary substituents selected from the following ⁇ group
  • the ⁇ group includes a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and a carbon number.
  • R is selected from a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms that may form a branch or a ring, and the ⁇ group.
  • An aryl group having 3 to 12 carbon atoms which may contain a hetero atom, or a carbon which may contain a hetero atom which may have one or more arbitrary substituents selected from the ⁇ group Represents an aralkyl group having an aryl moiety of 3 to 12;
  • R 1 and R 2 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, and a C 2 to 7 group that may form a branch or a ring.
  • N is an integer from 1 to 5
  • the present invention further provides a novel nuclease-resistant nucleic acid prepared by any of the methods described above.
  • high nuclease resistance can be imparted not only to an existing nucleic acid but also to a DNA strand of unknown sequence or a DNA strand having a long base length exceeding 100 residues very easily. Is possible. Therefore, it is useful in the field of medicines and diagnostics, in the field of genomic research, particularly in the preparation of reagents for genomic research.
  • Residual rate of single-stranded DNA (ODN1 # K, ODN1 # L, ODN1 # M, and ODN1 # Q) with a bridged nucleotide added to the 3 ′ end and an untreated single strand (ODN1) by nuclease treatment It is a graph which shows a time-dependent change.
  • Thrombin-binding aptamers (TBA1 # K, TBA1 # L, TBA1 # M, and TBA1 # Q) in which a bridging nucleotide is added to the 3 ′ end and the residual ratio of untreated thrombin-binding aptamers (TBA1) by nuclease treatment It is a graph which shows a time-dependent change.
  • Reaction solution in which thrombin was incubated with thrombin-binding aptamer (TBA1 # K, TBA1 # L, TBA1 # M, and TBA1 # Q) to which a bridging nucleotide was added at the 3 ′ end and untreated thrombin-binding aptamer (TBA1) Is an electrogram by capillary electrophoresis.
  • linear alkyl group having 1 to 6 carbon atoms refers to any linear alkyl group having 1 to 6 carbon atoms, specifically, a methyl group, an ethyl group, an n-propyl group, An n-butyl group, an n-pentyl group, or an n-hexyl group.
  • linear alkoxy group having 1 to 6 carbon atoms includes an alkoxy group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methyloxy group, an ethyloxy group, and an n-propyloxy group.
  • linear alkylthio group having 1 to 6 carbon atoms includes an alkylthio group having an arbitrary linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methylthio group, an ethylthio group, and an n-propylthio group.
  • a linear alkylamino group having 1 to 6 carbon atoms includes an alkylamino group having one or two alkylamino groups having any linear alkyl group having 1 to 6 carbon atoms. To do. Examples thereof include a methylamino group, a dimethylamino group, an ethylamino group, a methylethylamino group, and a diethylamino group.
  • an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring means any linear alkyl group having 1 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. It includes a chain alkyl group and any cyclic alkyl group having 3 to 7 carbon atoms.
  • arbitrary linear alkyl groups having 1 to 7 carbon atoms include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, and n-heptyl group.
  • Examples of the branched alkyl group having 3 to 7 carbon atoms include isopropyl group, isobutyl group, tert-butyl group, isopentyl group and the like, and optional cyclic alkyl group having 3 to 7 carbon atoms include A cyclobutyl group, a cyclopentyl group, a cyclohexyl group, etc. are mentioned.
  • an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring means any straight chain alkenyl group having 2 to 7 carbon atoms, any branch having 3 to 7 carbon atoms. It includes a chain alkenyl group and any cyclic alkenyl group having 3 to 7 carbon atoms.
  • the arbitrary branched chain alkenyl group having 3 to 7 carbon atoms there are isopropenyl group, 1-methyl-1-propenyl group, 1-methyl -2-propenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-methyl-2-butenyl group, etc.
  • any cyclic alkenyl group having 3 to 7 carbon atoms Includes a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and the like.
  • aryl group having 3 to 12 carbon atoms that may contain a hetero atom refers to any aromatic hydrocarbon having 6 to 12 carbon atoms and a ring structure that is composed of only hydrocarbons. Any heteroaromatic compound having 3 to 12 carbon atoms including an atom (nitrogen atom, oxygen atom, or sulfur atom) is included. Examples of the aromatic hydrocarbon having 6 to 12 carbon atoms composed of only hydrocarbons include a phenyl group, a naphthyl group, an indenyl group, an azulenyl group, and the like, and a 3 to 12 carbon atom having a hetero atom in the ring structure.
  • Arbitrary heteroaromatic compounds include pyridyl group, pyrrolyl group, quinolyl group, indolyl group, imidazolyl group, furyl group, thienyl group and the like.
  • aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a hetero atom examples include a benzyl group, a phenethyl group, a naphthylmethyl group, a 3-phenylpropyl group, -Phenylpropyl, 4-phenylbutyl, 2-phenylbutyl, pyridylmethyl, indolylmethyl, furylmethyl, thienylmethyl, pyrrolylmethyl, 2-pyridylethyl, 1-pyridylethyl, 3 -Thienylpropyl group and the like.
  • C2-C10 alkylene group or alkenylene group examples include methylene group, ethylene group, propylene group, pentenylene group, hexenylene group, vinylene group, propenylene group, butadienylene group and the like. It is done.
  • the method for preparing a nuclease-resistant nucleic acid of the present invention includes a step of mixing and incubating a nucleic acid consisting of at least three bases, a terminal deoxynucleotide transferase, and a 2 ', 4'-bridged nucleoside triphosphate.
  • the nucleic acid to which nuclease resistance is imparted may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the base length of the nucleic acid is not particularly limited as long as it is at least three base lengths.
  • the nucleic acid may be derived from any of nucleic acids isolated from natural products, recombinantly expressed nucleic acids, chemically synthesized nucleic acids, and enzymatically synthesized nucleic acids.
  • the nucleic acid may contain artificially modified bases and modified bases (such as methylation). Further, a labeling substance such as a fluorescent substance may be bound.
  • nucleic acids that can be suitable targets include various functional nucleic acids such as antisense DNA molecules, antigene DNA molecules, DNA aptamers, DNAzymes, and siRNA.
  • a nuclease refers to a nuclease that hydrolyzes a phosphodiester bond between a sugar of DNA or RNA and a phosphate to form nucleotides.
  • Nucleases are ubiquitous in vivo or in cells, so that when exogenous nucleic acid enters in vivo or in cells, it can be degraded by this enzyme.
  • nuclease resistance is imparted to the nucleic acid in order to prevent such degradation by nuclease.
  • nucleases examples include DNA-specific deoxyribonucleases, RNA-specific ribonucleases, and nucleases that act on both DNA and RNA. Also included are nucleases that act on single-stranded DNA, double-stranded DNA, single-stranded RNA, and DNA-RNA hybrids. In the present invention, resistance is preferably imparted to the exonuclease that degrades from the outside of the nucleic acid sequence, that is, from the 5 ′ end or 3 ′ end of the nucleic acid, more preferably to the exonuclease that degrades from the 3 ′ end. obtain.
  • the terminal deoxynucleotide transferase (TdT) used in the method of the present invention links a nucleotide having deoxyribose to the 3′-OH end of double-stranded and single-stranded DNA without depending on the template (addition). It is an enzyme that catalyzes the reaction.
  • TdT can also use a radiolabeled nucleotide or a nucleotide labeled with a hapten such as digoxigenin or biotin as a substrate.
  • the origin of TdT used in the present invention is not particularly limited, and commercially available TdT can be used.
  • TdT is typically 120 ⁇ L of reaction (120 mM potassium cacodylate, 1 mM CoCl 2 , 1 mM dTTP, 0.1 OD d (pT) 6 , and d (pT) 6 as a primer, and This is expressed as the amount of activity for incorporating 1 nmol of dAMP into the acid precipitate when reacted at 37 ° C. for 30 minutes in 6.25 pmol [ 3 H] -dTTP).
  • the 2 ', 4'-bridged nucleoside triphosphate used in the method of the present invention is represented by the following formula I, formula II or formula III:
  • Base represents a purin-9-yl group or a 2-oxo-1,2-dihydropyrimidin-1-yl group which may have one or more arbitrary substituents selected from the following ⁇ group
  • the ⁇ group includes a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and a carbon number.
  • R is selected from a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms that may form a branch or a ring, and the ⁇ group.
  • An aryl group having 3 to 12 carbon atoms which may contain a hetero atom, or a carbon which may contain a hetero atom which may have one or more arbitrary substituents selected from the ⁇ group Represents an aralkyl group having an aryl moiety of 3 to 12;
  • R 1 and R 2 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, and a C 2 to 7 group that may form a branch or a ring.
  • Base is a purine base (ie, purin-9-yl group) or pyrimidine base (ie, 2-oxo-1,2-dihydropyrimidin-1-yl group).
  • bases are a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and 1 to carbon atoms. It may have one or more arbitrary substituents selected from the ⁇ group consisting of 6 linear alkylamino groups and halogen atoms.
  • the base include a 6-aminopurin-9-yl group (adenylyl group), a 2,6-diaminopurin-9-yl group, and 2-amino-6-chloropurin-9-yl.
  • 2-amino-6-fluoropurin-9-yl group 2-amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurin-9-yl group (guaninyl group)
  • Base is the following structural formula in that it can be a more suitable substrate for TdT:
  • 2,4-dihydroxy-5-methylpyrimidin-1-yl group (thyminyl group), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group (cytosynyl group), A 6-aminopurin-9-yl group (adenylyl group) and a 2-amino-6-hydroxypurin-9-yl group (guaninyl group) are preferable, and in particular, 2,4-dihydroxy-5-methylpyrimidine- A 1-yl group (thyminyl group) is preferred.
  • R is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, an alkenyl group having 2 to 7 carbon atoms which may form a branch or a ring, an acyl group optionally having one or more optional substituents selected from the ⁇ group, a sulfonyl group having an optional substituent selected from the ⁇ group, and an optional substituent selected from the ⁇ group
  • R is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group, and more preferably, R is a hydrogen atom or a methyl group.
  • R 1 and R 2 are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms that may form a branch or a ring, and a carbon that may form a branch or a ring.
  • n is an integer of 1 to 5, preferably 1 or 2, and more preferably 1.
  • m is an integer of 1 to 5, preferably 1 or 2, and more preferably 1.
  • Base is a thyminyl group
  • R is a hydrogen atom
  • R 1 and R Structural formulas (wherein LTP, MTP, and QTP, respectively) are shown below when 2 is a hydrogen atom and a phenyl group, and m and n are both 1, respectively.
  • Such 2 ′, 4′-bridged nucleoside triphosphates can be converted from any nucleoside (natural or non-bridged) to 2 ′, 4′-bridged by the method described in Non-Patent Documents 1 to 4 or 6.
  • a nucleoside can be prepared and further prepared by triphosphorylation according to the method described in Non-Patent Document 5.
  • a commercially available 2 ', 4'-bridged nucleoside may be used.
  • a nucleic acid consisting of at least three bases, terminal deoxynucleotide transferase (TdT), and 2 ′, 4′-bridged nucleoside triphosphate are mixed and incubated, whereby a nuclease resistant nucleic acid is obtained.
  • TdT terminal deoxynucleotide transferase
  • 2 ′, 4′-bridged nucleoside triphosphate are mixed and incubated, whereby a nuclease resistant nucleic acid is obtained.
  • the amount of 2 ′, 4′-bridged nucleoside triphosphate with respect to the nucleic acid to be imparted with resistance is not particularly limited, but it is excessive in order to efficiently and reliably add to the 3 ′ end of the nucleic acid. It is preferable that Further, the amount of TdT used is not particularly limited, and can be appropriately set according to the amount of nucleic acid.
  • the composition of the reaction solution is not particularly limited as long as the activity of TdT is exhibited. When using commercially available TdT, it is preferable to use the buffer solution attached to TdT.
  • the incubation conditions are not particularly limited as long as they are conditions that are usually suitable for exerting the activity of TdT, and are appropriately set according to the TdT used.
  • the incubation temperature can be 30 ° C. to 40 ° C., preferably 37 ° C.
  • the incubation time is also appropriately set according to the amount of TdT, the amount of nucleic acid and 2 ', 4'-crosslinked nucleoside triphosphate.
  • any easily-obtained TdT and 2 ′, 4′-bridged nucleoside triphosphates are incubated under the conditions commonly used by those skilled in the art to achieve any desired
  • a 2 ′, 4′-bridged nucleotide can be added to the 3 ′ end of the nucleic acid.
  • 2 ', 4'-crosslinked nucleoside triphosphate and TdT may be provided in the form of a kit.
  • a kit may also contain a buffer solution, instructions for use, etc. suitable for performing the above incubation.
  • nucleic acid to which these 2 ', 4'-bridged nucleotides are added is a novel nucleic acid to which resistance against nucleases has been imparted. Therefore, the desired nucleic acid function can be exhibited more effectively in vivo where nuclease is present.
  • KTP was prepared according to the description of Non-Patent Document 5.
  • 5-methyl-2′-O, 4′-C-methyleneuridine (nucleoside 1: synthesized according to Non-Patent Document 6) (100 mg, 0.370 mmol) and N, N, N ′, N′-tetramethyl- 1,8-Naphthalenediamine (Proton® Sponge®: 119 mg; 0.555 mmol; 1.5 eq) was placed in the flask and dried overnight under vacuum.
  • Trimethyl phosphoric acid (2.6 mL) was then placed in the flask under an argon atmosphere and the mixture was cooled to 0 ° C.
  • the residue was purified using a Sephadex® DEAE A-25 column with a linear gradient of 0.05 to 1.0 M triethylammonium bicarbonate buffer (pH 8). Corresponding fractions were combined and evaporated under reduced pressure. To remove excess pyrophosphate, the residue was purified by reverse phase MPLC with a linear gradient from 0% (v / v) to 20% (v / v) acetonitrile in 10 mM triethylammonium acetate buffer, pH 7. .
  • LTP was prepared according to the description of Non-Patent Document 5. Except that 5-methyl-2′-O, 4′-C- (methyleneoxymethylene) uridine (nucleoside 2: synthesized according to Non-Patent Document 2) (106 mg, 0.353 mmol) was used in place of nucleoside 1, A cross-linked nucleoside triphosphate LTP was obtained by the same procedure as in Reference Preparation Example 1 (18.7 ⁇ mol: yield 5.3%).
  • MTP was prepared according to the description in Non-Patent Document 5.
  • triethylamine hydrogen trifluoride (313 ⁇ L; 1.91 mmol; 5 equivalents) was added to a THF solution (4.1 mL) of nucleoside 3 (synthesized according to Non-Patent Document 4) (201 mg; 0.381 mmol) with stirring. .
  • the reaction mixture was stirred at room temperature for 3 hours. After evaporation, the residue was purified by reverse phase column chromatography (0% (v / v) to 5% (v / v) aqueous acetonitrile).
  • the residue containing nucleoside 4 was dissolved in methanol and crystallized to give nucleoside 4 in quantitative yield (108 mg; 0.379 mmol).
  • nucleoside 4 (57.8 mg, 0.203 mmol) was used instead of nucleoside 1 (3.19 ⁇ mol). : Yield 1.6%).
  • Example 1 Addition reaction of bridged nucleotide to DNA 3 'end by terminal deoxynucleotide transferase (TdT)) 26mer single-stranded DNA (ggcgttgagtgagtgaatgagtgagt: SEQ ID NO: 1) (ODN1; 0.4 ⁇ M) (purchased from Nippon Bioservice Co., Ltd.), terminal deoxynucleotide transferase (TdT; 0.125, 0.150 or 0.175 unit / ⁇ L) (purchased from Takara Bio Inc.), buffer solution (1 ⁇ ) attached to this enzyme and 200 ⁇ M of the crosslinked nucleoside triphosphate obtained in Preparation Examples 1 to 3 above 20 ⁇ L of each reaction solution containing (KTP, LTP or MTP) was prepared and incubated at 37 ° C.
  • TdT terminal deoxynucleotide transferase
  • DNA having a slightly higher molecular weight that is, DNA to which a cross-linked nucleotide is added can be obtained. I understood it. It was also found that this added DNA was generated in a dose-dependent manner with the enzyme TdT.
  • nucleoside 6 was synthesized from nucleoside 5.
  • Zinc chloride 114 mg; 0.838 mmol
  • an anhydrous benzaldehyde solution 3.7 mL
  • saturated aqueous sodium hydrogen carbonate was added, and the mixture was extracted with ethyl acetate.
  • the organic layer was washed with water and saturated brine, and dried over anhydrous sodium sulfate.
  • nucleoside 7 (338 mg: 78% yield) as a white foam. Obtained as material.
  • tetrabutylammonium fluoride (1.0 M in tetrahydrofuran; 0.0712 ml; 0.0712 mmol) was added to a tetrahydrofuran solution (0.9 mL) of nucleotide 7 (22.0 mg; 0.0356 mmol), and the mixture was cooled with ice. Stir for 1 hour. After the solvent was distilled off, the residue was purified by silica gel chromatography (ethyl acetate) to obtain nucleoside 8 (12.9 mg: yield 96%) as a white solid.
  • nucleoside 8 106 mg; 0.282 mmol
  • nucleoside 1 6.580 ⁇ mol: Yield 2.3%
  • Example 2 Evaluation of nuclease resistance of single-stranded DNA having a bridged nucleotide added to the 3 'end
  • Single-stranded DNA added with cross-linked nucleotides KTP, LTP, MTP, and QTP at the 3 ′ end in the same procedure as in Example 1 (referred to as ODN1 # K, ODN1 # L, ODN1 # M, and ODN1 # Q, respectively)
  • ODN1 # K, ODN1 # L, ODN1 # M, and ODN1 # Q respectively
  • ODN1 # K aqueous solution containing any one single-stranded DNA
  • 2 ⁇ L of an aqueous solution containing snake venom phosphodiesterase I 25 ⁇ unit / ⁇ L
  • a reaction solution (5 ⁇ L) was prepared by mixing 1 ⁇ L of a reaction buffer solution (250 mM Tris-HCl, 50 mM MgCl 2 , pH 8.0), incubated at 37 ° C
  • a dye solution (0.1% (v / v) bromophenol blue) and a 7M urea solution (3 mM EDTA) were added to the removed reaction solution, and then heat-denatured at 94 ° C., and 20% (w / v).
  • Electrophoresis was performed using a polyacrylamide denaturing gel (denaturing PAGE; 300 V, 49 ° C., 130 minutes). After electrophoresis, the gel was visualized by laser irradiation (488 nm) using a molecular imager (manufactured by BioRad). From the band intensity in the obtained gel photograph (FIG. 2), the ratio (%) of unreacted single-stranded DNA was calculated and plotted against the reaction time (FIG. 3).
  • Example 3 Evaluation of nuclease resistance of thrombin-binding DNA aptamer having a bridging nucleotide added to the 3 'end
  • TBA Thrombin Binding Aptamer, purchased from Sigma-Aldrich
  • the thrombin-binding DNA aptamer (agtccgtggtagggcaggttggggtgact: SEQ ID NO: 2) used was labeled with carboxyfluorescein at the 5 ′ end.
  • TBA1 # K, TBA1 # L, TBA1 # M, and TBA1 # Q thrombin-binding DNA aptamers
  • TBA1 # Q unmodified thrombin-binding DNA aptamers
  • a 5 ⁇ concentration reaction buffer 250 mM Tris-HCl, 50 mM MgCl 2 , 2.5% (w / v) Tween 20, pH 8.0
  • a reaction solution 5 ⁇ L was prepared, incubated at 37 ° C., 10 minutes after starting the reaction, 20 minutes after, 30 After 60 minutes and 120 minutes, the reaction solution was taken out.
  • a dye solution (0.1% (v / v) bromophenol blue) and a 7M urea solution (9 mM EDTA) were added to the removed reaction solution, and then heat-denatured at 94 ° C., and 20% (w / v).
  • Electrophoresis was performed using a polyacrylamide denaturing gel (denaturing PAGE; 300 V, 49 ° C., 130 minutes). After electrophoresis, the gel was visualized by laser irradiation (488 nm) using a molecular imager (BioRad). The ratio (%) of unreacted single-stranded DNA was calculated from the band intensity in the obtained gel photograph and plotted against the reaction time. The results are shown in FIG.
  • DNAs treated with cross-linked nucleotides LTP, MTP, and QTP and TdT are all untreated TBA1 and TBA # K.
  • the residual rate was higher than that of the exonuclease (snake venom phosphodiesterase I).
  • the residual rate of DNAs (TBA1 # M and TBA1 # Q) in which bridged nucleotides were added to the 3 'end using MTP and QTP was high.
  • Example 4 Evaluation of the stability in serum of a thrombin-binding DNA aptamer having a bridged nucleotide added to the 3 'end
  • Thrombin-binding DNA aptamers (TBA1 # K, TBA1 # L, TBA1 # M, and TBA1 # Q) and a non-modified thrombin-binding DNA aptamer with a bridged nucleotide added to the 3 ′ end prepared in the same manner as in Example 3 above 0.5 ⁇ L of an aqueous solution (4 ⁇ M) containing any one DNA aptamer of (TBA1), 4 ⁇ L of serum (human male type AB plasma: Sigma-Aldrich), and a 10 ⁇ concentration reaction buffer (500 mM) (5 ⁇ L) prepared by mixing 0.5 ⁇ L of Tris-HCl, 100 mM MgCl 2 , pH 8.0) and incubating at 37 ° C., 10 minutes, 20 minutes, and 30 minutes after the start
  • reaction solution was taken out.
  • a dye solution (0.1% (v / v) bromophenol blue) and a 7M urea solution (3 mM EDTA) were added to the taken out reaction solution, and then heat-denatured at 94 ° C. to obtain 20% (w / v).
  • Electrophoresis was performed using a polyacrylamide denaturing gel (denaturing PAGE; 300 V, 49 ° C., 130 minutes). After electrophoresis, the gel was visualized by laser irradiation (488 nm) using a molecular imager (BioRad). The ratio (%) of unreacted single-stranded DNA was calculated from the band intensity in the obtained gel photograph and plotted against the reaction time. The results are shown in FIG.
  • Example 5 Evaluation of binding affinity of thrombin-binding DNA aptamer with a bridging nucleotide added to the 3 ′ end to thrombin
  • Thrombin-binding DNA aptamers (TBA1 # K, TBA1 # L, TBA1 # M, and TBA1 # Q) with a bridged nucleotide added to the 3 ′ end and the unmodified thrombin-binding DNA aptamer prepared in the same manner as in Example 3 above
  • the electrophoresis conditions were as follows.
  • a BECKMAN COULTER, P / ACE TM MDQ apparatus was used for capillary electrophoresis.
  • the cartridge temperature was set at 25 ° C and the sample storage at 15 ° C.
  • Detection was performed with a fluorescence detector, and the excitation wavelength was set to 488 nm and the monitor wavelength was set to 520 nm.
  • the capillary used was a capillary having an inner diameter of 75 ⁇ m (manufactured by BECKMAN COULTER) adjusted to a length of 30.2 cm (the length to the window was 20 cm).
  • As the running buffer 0.4% (w / v) borate buffer (0.3% (w / v) sodium borate, pH 8.35: manufactured by BECKMAN COULTER) was used.
  • the peak with a migration time of around 2 minutes was a complex of each TBA and thrombin, and the peak with a migration time of around 3 minutes was a free form of each TBA.
  • TBA1 # M a third peak appeared between the complex and the free form (around 2.6 minutes). This is because the free TBA1 # M dissociated from thrombin during the migration was complex. This is thought to be due to some higher order structure due to the interaction with the body. Therefore, this peak was regarded as a complex.
  • the dissociation constant Kd was calculated from the electrogram by the following equation as described in M.V. Berezovski et al., Nature Protocols, 2006, Vol. 1, No. 3, page 1362. The calculated Kd is shown in Table 1 below.
  • the enzymatic addition of cross-linked nucleotides to various functional nucleic acids facilitates the nuclease resistance of those nucleic acid molecules. It is possible to improve it. Therefore, it is useful for genome technologies (antisense method, antigene method, RNAi, decoy method, gene homologous recombination, ribozyme, DNA enzyme, etc.) targeting RNA, single-stranded DNA, and double-stranded DNA as molecular targets. . Therefore, it can be used for the development and preparation of genomic research reagents such as basic biochemical research tools, pharmaceuticals, diagnostics, and analytical reagents as common base materials for nucleic acid drug development and diagnostic systems.
  • genomic research reagents such as basic biochemical research tools, pharmaceuticals, diagnostics, and analytical reagents as common base materials for nucleic acid drug development and diagnostic systems.

Abstract

L'invention porte sur un procédé de préparation d'un acide nucléique résistant aux nucléases, qui comprend les étapes consistant à mélanger un acide nucléique composé d'au moins trois nucléotides, une transférase de désoxynucléotide terminal et un nucléoside triphosphate 2',4'-réticulé l'un avec l'autre et l'incubation du mélange résultant. Des exemples du nucléoside triphosphate 2',4'-réticulé devant être utilisé dans le procédé comprennent le 5-méthyl-2'-O,4'-C-(méthylèneoxy-méthylène)uridine-5'-triphosphate, le 5-méthyl-2'-O,4'-C-(aminométhylène)uridine-5'-triphosphate et le 5-méthyl-2'-O,4'-C-(benzyloxyméthylène)uridine-5'-triphosphate. Conformément au procédé, il devient possible de conférer une excellente résistance aux nucléases à un acide nucléique fonctionnel existant après cela d'une manière simple.
PCT/JP2009/064522 2008-08-22 2009-08-19 Nouveau dérivé d'acide nucléique et procédé de préparation d'un acide nucléique résistant aux nucléases par son utilisation WO2010021344A1 (fr)

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WO2010113937A1 (fr) * 2009-03-31 2010-10-07 武田薬品工業株式会社 Procédé de production de nucléoside
EP2447273A1 (fr) * 2009-06-23 2012-05-02 Takeda Pharmaceutical Company Limited Méthode de synthèse d'un acide nucléique
WO2014112463A1 (fr) * 2013-01-15 2014-07-24 国立大学法人大阪大学 Nucléoside et nucléotide ayant une structure de sulfonamide
WO2014126229A1 (fr) * 2013-02-18 2014-08-21 塩野義製薬株式会社 Nucléoside et nucléotide possédant une structure hétérocyclique azotée
WO2022065413A1 (fr) * 2020-09-25 2022-03-31 株式会社理研ジェネシス Nouvel acide nucléique artificiel, son procédé de production et son utilisation

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010113937A1 (fr) * 2009-03-31 2010-10-07 武田薬品工業株式会社 Procédé de production de nucléoside
US8653254B2 (en) 2009-03-31 2014-02-18 Takeda Pharmaceutical Company Limited Process for producing nucleoside
EP2447273A1 (fr) * 2009-06-23 2012-05-02 Takeda Pharmaceutical Company Limited Méthode de synthèse d'un acide nucléique
EP2447273A4 (fr) * 2009-06-23 2013-06-19 Takeda Pharmaceutical Méthode de synthèse d'un acide nucléique
WO2014112463A1 (fr) * 2013-01-15 2014-07-24 国立大学法人大阪大学 Nucléoside et nucléotide ayant une structure de sulfonamide
JPWO2014112463A1 (ja) * 2013-01-15 2017-01-19 国立大学法人大阪大学 スルホンアミド構造を有するヌクレオシドおよびヌクレオチド
WO2014126229A1 (fr) * 2013-02-18 2014-08-21 塩野義製薬株式会社 Nucléoside et nucléotide possédant une structure hétérocyclique azotée
US9315535B2 (en) 2013-02-18 2016-04-19 Shionogi & Co., Ltd. Nucleoside and nucleotide having nitrogen-containing heterocycle structure
JPWO2014126229A1 (ja) * 2013-02-18 2017-02-02 塩野義製薬株式会社 含窒素複素環構造を有するヌクレオシド及びヌクレオチド
WO2022065413A1 (fr) * 2020-09-25 2022-03-31 株式会社理研ジェネシス Nouvel acide nucléique artificiel, son procédé de production et son utilisation

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