WO2023210767A1 - Oligonucleotide chemical ligation reaction - Google Patents

Oligonucleotide chemical ligation reaction Download PDF

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WO2023210767A1
WO2023210767A1 PCT/JP2023/016716 JP2023016716W WO2023210767A1 WO 2023210767 A1 WO2023210767 A1 WO 2023210767A1 JP 2023016716 W JP2023016716 W JP 2023016716W WO 2023210767 A1 WO2023210767 A1 WO 2023210767A1
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oligonucleotide
variant
nucleic acid
group
linked
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PCT/JP2023/016716
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French (fr)
Japanese (ja)
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康雄 小松
直 小島
悠 平野
葉玲 櫻井
恵美 斎藤
浩一 南海
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国立研究開発法人産業技術総合研究所
株式会社ジーンデザイン
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Publication of WO2023210767A1 publication Critical patent/WO2023210767A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • 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
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • C07H21/02—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 ribosyl as saccharide radical
    • 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
    • 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

Definitions

  • the present disclosure relates to chemical ligation reactions for the synthesis of long oligonucleotides or polynucleotides or variants thereof and their applications.
  • RNA molecules are difficult to chemically synthesize, they are often produced by a transcription reaction using RNA synthase.
  • RNA synthase it is difficult to site-specifically introduce chemically modified nucleic acids.
  • Modified nucleic acids are essential for increasing resistance to nucleolytic enzymes in vivo, exhibiting high affinity with proteins, and imparting new functions to RNA, such as labeling with fluorescent molecules.
  • oligonucleotides or polynucleotides (herein, typically referred to as "oligonucleotides”; when referred to as oligonucleotides, it is understood that there is no particular limitation in length) or variants thereof. Concerning connection methods.
  • a 2'-fluoro-5'-amino-nucleic acid (2F-5N nucleic acid) having a fluorine atom at the 2' position of a 5'-amino-nucleic acid (5N nucleic acid) is synthesized.
  • the 2F-5N nucleic acid can be stably bound to the template nucleic acid by 2'-fluoro modification, and the reactivity of the 5'-amino group can be improved. We can expect improvement.
  • fluorine atoms have hydrogen bonds like the 2' hydroxyl groups of nucleic acids, they are suitable for interaction with proteins, and have high resistance to nucleolytic enzymes, so they can have a synergistic effect when combined with amino groups in ligation reactions. It will be done.
  • the present disclosure makes it possible to chemically bind chemically modified RNA molecules with high efficiency, making it possible to create long-chain RNAs with new functions (as a representative diagram, 1). Furthermore, since this reaction is applicable not only to RNA but also to synthetic DNA, it provides an effective technique for producing functional DNA.
  • the present invention provides, for example, the following items.
  • a method for linking a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof comprising: arranging the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof under conditions in which linkage occurs; A linkage occurs between the 3' end of the first oligonucleotide or its variant and the 5' end of the second oligonucleotide or its variant, and the terminal monomer portion on the 5' end side of the second oligonucleotide is containing at least one modification; Method.
  • (Item 2) The method according to any one of the preceding items, wherein the 3' end of the first oligonucleotide or variant thereof comprises a phosphate group or a thiophosphate group.
  • (Item 3) The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof comprises an optionally substituted amino group.
  • (Item 4) The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof comprises an optionally substituted amino group at the 5' position of its terminal monomer moiety.
  • (Item 5) A method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof comprises an unsubstituted amino group at the 5' position of its terminal monomer moiety.
  • (Item 6) A method according to any of the preceding items, wherein the amino group is substituted by a C 1-6 alkyl group.
  • (Item 7) A method according to any one of the preceding items, wherein said amino group is substituted by a methyl group or an ethyl group.
  • (Item 8) The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof is substituted with halo or -OH at the 2' position of its terminal monomer moiety.
  • (Item 16) The method according to any one of the preceding items, wherein the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof are linked via a phosphate group or a thiophosphate group. .
  • (Item 17) The method of any one of the preceding items, wherein the conditions under which the ligation occurs includes using a template nucleic acid.
  • the template nucleic acid has a sequence and chain length that are continuous and complementary to the first oligonucleotide or its variant and the second oligonucleotide or its variant. the method of.
  • (Item 19) The method according to any one of the preceding items, wherein the template nucleic acid has a chain length of 10 residues or more.
  • (Item 20) The method according to any one of the preceding items, wherein the template nucleic acid has a chain length of 20 to 40 residues.
  • (Item 21) The template nucleic acid is not linked to the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof, or either one of the oligonucleotides or a variant thereof, or both.
  • a method according to any one of the preceding items, wherein the method is linked to an oligonucleotide or a variant thereof.
  • the sequence of the template nucleic acid includes a sequence complementary to the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof, or mismatched base pairs within a range that does not prevent the formation of double strands. , the method described in any one of the preceding items.
  • a method according to any of the preceding items, wherein the conditions under which the ligation occurs includes the use of an activator of the oligonucleotide ligation reaction.
  • the first oligonucleotide or its variant has the following formula (I) (In the formula, R - is S - or O - , Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine) , 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-amino purine, or 2,6-diaminopurine, A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, n is an integer greater than or equal to 1, and R 1 is a hydroxyl group at the 5'-position.
  • the second oligonucleotide or its variant has the following formula (II) (wherein, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, p is an integer greater than or equal to 1, and R 2 is a nucleic acid in which the 3′-position is a hydroxyl group, a phosphate group, or a thiophosphoric acid group, or a nucleic acid derivative)
  • the method according to any one of the preceding items, wherein the compound is a compound of the formula: (Item 28) A method according to any one of the preceding items, wherein said X is a fluoro group.
  • (Item 29) A method according to any one of the preceding items, wherein p is an integer from 5 to 1000.
  • (Item 30) A method according to any one of the preceding items, wherein the spacer is an optionally substituted C 1-10 alkylene or PEG.
  • (Item 31) A method according to any one of the preceding items, wherein the spacer is a C3 alkylene.
  • the activating agent is a carbodiimide condensing agent, a phosphonium condensing agent, a uronium condensing agent or a triazine condensing agent.
  • the carbodiimide condensing agent is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC), diisopropylcarbodiimide (DIPCI) or dicyclohexylcarbodiimide (DCC). the method of.
  • the phosphonium condensing agent is bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), 1H-benzotriazol-1-yloxytris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyBOP), 6-trifluoro Methyl-1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyFOP), 1H-benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) or (7-aza Benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP).
  • PyBroP bromotripyrrolidinophosphonium hexafluorophosphate
  • PyBOP 1H-benzotriazol-1-yloxy
  • the uronium-based condensing agent is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazole-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroboric acid salt (TSTU) or ⁇ [(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy ⁇ -4-morpholinomethylene ⁇ dimethylammonium hexafluorophosphate (COMU).
  • HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluoro
  • the activator may be 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt) or N- A method according to any one of the preceding items, used in combination with hydroxysuccinimide (HOSu).
  • oligonucleotides or variants thereof in which the 5' end of the first oligonucleotide or variant thereof and the 3' end of the second oligonucleotide or variant thereof, and the first oligonucleotide or variant thereof;
  • the oligonucleotide or variant thereof to which the 5' end of the first oligonucleotide or variant thereof and the template nucleic acid are linked is linked to the second oligonucleotide or variant thereof.
  • a composition for producing a linked oligonucleotide or a variant thereof using a nucleotide or a variant thereof, or the first oligonucleotide or a variant thereof. (Item 46) For producing a linked oligonucleotide using a template nucleic acid comprising the first oligonucleotide according to item 1 or a variant thereof, the second oligonucleotide according to item 1 or a variant thereof, or a combination thereof. Composition.
  • a kit for linking oligonucleotides or variants thereof comprising: a first oligonucleotide according to any one of the preceding items or a variant thereof; a second oligonucleotide according to any one of the preceding items or a variant thereof; an activator; A kit comprising a template nucleic acid.
  • (Item 48) Producing a linked oligonucleotide comprising a first oligonucleotide according to any one of the preceding items or a variant thereof and a second oligonucleotide according to any one of the preceding items or a variant thereof Raw material composition for.
  • (Item 49) The raw material composition according to any one of the preceding items, wherein the raw material for producing the second oligonucleotide or a variant thereof contains a nucleoside having a fluoro group at the 2' position and an amino group at the 5' position. thing.
  • (Item 50) Containing raw materials (monomers or substituent introduction reagents, etc.) for the first and/or second oligonucleotides or variants thereof; A composition for producing a first oligonucleotide according to any one of the preceding items or a variant thereof and/or a second oligonucleotide according to any one of the preceding items or a variant thereof. thing.
  • the raw material for the first and/or second oligonucleotide or a variant thereof is an amidide or a deoxynucleoside, or a deoxynucleoside in which the 3' position is a hydroxyl group. Composition of.
  • Y is a hydrogen group or an alkoxy group
  • B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine , 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine.
  • X is a hydroxy group or a halogen
  • A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative
  • n is an integer of 1 or more
  • p is an integer of 1 or more
  • R 1 is a nucleic acid or a nucleic acid derivative in which the 5'-position is a hydroxyl group
  • R 1 is a nucleic acid or a nucleic acid derivative in which the 2'-position is a hydrogen group or an alkoxy group and the 5'-position is an amino group.
  • R 2 is a nucleic acid or a nucleic acid derivative whose 3'-position is a hydroxyl group, phosphate group, or thiophosphoric acid group, or R 2 is a hydroxyl group or a halogen at the 2'-position and a phosphoric acid group at the 3'-position.
  • R 1 and R 2 may be taken together to form a single bond, and R 1 and R 2 are R 1 and R 2 of different compounds.
  • 2 and R 3 is an optionally substituted amino group.
  • Said Y is a methoxy group, and said B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine. , pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine, and the X is , a fluoro group, and R 1 and R 2 are taken together to form a single bond.
  • (Item 56) A compound according to any one of the preceding items, wherein n is an integer from 5 to 1000 and p is an integer from 5 to 1000.
  • (Item 57) A compound according to any one of the preceding items, wherein R 3 is an unsubstituted amino group.
  • (Item 58) A compound according to any one of the preceding items, wherein R 3 is an amino group substituted by a C 1-6 alkyl group.
  • (Item 59) A compound according to any one of the preceding items, wherein R 3 is an amino group substituted by a methyl group or an ethyl group.
  • (Item 60) A compound according to any one of the preceding items, wherein B is each independently adenine, cytosine, methylcytosine, guanine, thymine, or uracil.
  • (Item 61) A compound according to any one of the preceding items, wherein said R 1 and said R 2 are taken together to form a single bond.
  • (Item 62) The 5' end of the first oligonucleotide or variant thereof according to any one of the preceding items and the 3' end of the second oligonucleotide or variant thereof according to any one of the preceding items.
  • a linked oligonucleotide or a variant thereof in which a plurality of linked oligonucleotides or variants thereof, the first oligonucleotide or a variant thereof, and the second oligonucleotide or a variant thereof are linked. . (Item 63) The 5' end of the first oligonucleotide or variant thereof according to any one of the preceding items and the oligonucleotide or variant thereof linked to the template nucleic acid according to any one of the preceding items, A linked oligonucleotide or a variant thereof, which is linked to a second oligonucleotide or a variant thereof according to any one of the items.
  • (Item 64) The 3' end of the second oligonucleotide or variant thereof according to any one of the preceding items and the oligonucleotide or variant thereof linked to the template nucleic acid according to any one of the preceding items, A linked oligonucleotide or a variant thereof, which is linked to the oligonucleotide or variant thereof according to any one of the items.
  • (Item 65) The 5' end of the first oligonucleotide or variant thereof according to any one of the preceding items and the 3' end of the second oligonucleotide or variant thereof according to any one of the preceding items.
  • a linked oligonucleotide or a variant thereof, the 3' end of the first oligonucleotide or variant thereof, and the 5' end of the second oligonucleotide or variant thereof are linked. Nucleotides or variants thereof.
  • FIG. 1 shows a schematic diagram of the oligonucleotide ligation reaction.
  • the M at the 3' end of the 5' oligo and the Q at the 5' end of the 3' oligo are linked to link the two oligo chains.
  • Oligo may be RNA, DNA, or a mixture thereof.
  • FIG. 2 shows a reaction scheme of oligonucleotide ligation reaction.
  • an oligo with Y methoxy group, hydrogen, or fluoro group as the 2' position in the nucleotide residue of M
  • an oligo with X hydroxyl group, fluoro group as the 2' position in the nucleotide residue of Q.
  • a base oligonucleotide chain is used.
  • FIG. 3 shows the sequences of oligonucleotides or nucleic acids used in oligonucleotide ligation reactions.
  • FIG. 4 shows a synthesis scheme of a reagent (5'-amino-2'-fluoro-2',5'-dideoxyuridine amidite reagent) for introducing 2'F-5'-NH 2 -uridine into Q.
  • FIG. 5 shows the structure of a reagent (5'-amino-5'-deoxyuridine amidite reagent) for introducing 2'OH-5'-NH 2 -uridine into Q.
  • Figure 6 shows HPLC analysis of the reaction between 2Fps and DNFB.
  • FIG. 7 shows PAGE analysis of the reaction between DNFB-modified 5'-oligo and 3'-oligo.
  • FIG. 8 shows a comparison of reaction rates in a 120 min reaction between DNFB-modified 5'-oligo and 3'-oligo.
  • FIG. 9 shows PAGE analysis of a 6-hour reaction of 5'-oligo and 3'-oligo in the presence of DNFB.
  • FIG. 10 shows the reaction of 5'-oligo and 3'-oligo in the presence of EDC.
  • A Comparison of reaction rates in 60 min reaction of 5'oligo and 3'oligo
  • B HPLC analysis of 2OH5N and 2Mepo conjugate
  • C HPLC analysis of 2F5N and 2Mepo conjugate.
  • FIG. 11 shows a schematic diagram of a ligation reaction in which each strand serves as a template without using template DNA.
  • Figure 12 shows the synthesis of a reagent (5'-amino-2'-fluoro - 2',5'-dideoxyuridine amidite reagent, compound 5) for introducing 2'-F-5'-NH2cytidine into Q. Show the scheme.
  • Figure 13 shows the structure of a reagent for introducing 2'-OH-5'-NH 2 cytidine into Q (5'-amino-5'-deoxycytidine amidite reagent, compound 11).
  • FIG. 14 shows PAGE analysis of a 6-hour reaction of 5'-oligo and 3'-oligo in the presence of DNFB. A comparison of 2OH5N_C and 2F5N_C reactions using RNA and DNA templates is shown.
  • FIG. 15 shows a comparison of the reaction rates of 5'-oligo and 3'-oligo in a 30 min reaction in the presence of EDC.
  • group means a monovalent group unless otherwise specified. Examples of non-monovalent groups include alkylene groups (divalent) and the like. Furthermore, in the following description of substituents, etc., the term “group” may be omitted in some cases.
  • the number of substituents when defined as “optionally substituted” or “substituted” is not particularly limited as long as it is substitutable, unless there is a particular limitation; or more than one. Further, unless otherwise specified, the description of each substituent also applies when the substituent is a part of or a substituent of another substituent.
  • substituted upward refers to substitution such that the bond is directed upward with respect to the ring to which it is bonded.
  • nucleic acids nucleosides
  • furanose ring 5-membered ring
  • it is substituted on the same side as the nucleobase.
  • substituted downward refers to substitution such that the bond is directed downward with respect to the ring to which it is bonded.
  • nucleic acids nucleosides
  • any part of the group may be substituted.
  • “optionally substituted arylalkyl” and “substituted arylalkyl” may be substituted at the aryl moiety or substituted at the alkyl moiety, and include both the aryl moiety and the alkyl moiety. may be replaced.
  • C 1-6 means having 1 to 6 carbon atoms.
  • C 1-4 means that the number of carbon atoms is 1 to 4
  • C 1-3 means that the number of carbon atoms is 1 to 3. means.
  • descriptions in which the number of carbon atoms is limited are only preferred numerical ranges, and the present disclosure is intended to include groups having substituents with a number of carbon atoms other than the specified number of carbon atoms within the scope of the present disclosure. .
  • halogen atom refers to an atom belonging to the halogen group, such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. Preferably it is a fluorine atom or a chlorine atom. More preferably, it is a fluorine atom.
  • a "halogen atom” is sometimes referred to as “halogen” or "halo.”
  • hydroxyl group is a monovalent group of -OH. This group is sometimes referred to as a "hydroxy group” or "hydroxy.”
  • amino refers to the group -NH2 .
  • phosphate group is refers to The oxygen atom of the phosphoric acid group may be replaced with a sulfur atom, and a phosphoric acid group in which the oxygen atom is replaced with a sulfur atom is referred to herein as a "thiophosphoric acid group.”
  • the number of oxygen atoms to be substituted may be 1 to 4, but preferably 1.
  • alkyl refers to a straight or branched saturated aliphatic hydrocarbon group.
  • C 1-12 alkyl is an alkyl group having 1 to 12 carbon atoms, and examples thereof include C 1-6 alkyl, heptyl, isoheptyl, octyl, isooctyl, nonyl, isononyl, decyl, isodecyl, undecyl, isoundecyl. , dodecyl, isododecyl and the like, but are not limited to these.
  • C 1-6 alkyl is an alkyl group having 1 to 6 carbon atoms, and preferred examples include “C 1-4 alkyl", more preferably “C 1-3 alkyl", More preferred is “C 1-2 alkyl”. Specific examples of “C 1-4 alkyl” include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, and the like.
  • C 1-6 alkyl include, but are not limited to, C 1-4 alkyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, n-hexyl, etc. Not done.
  • alkoxy is a monovalent group of -O-alkyl.
  • Preferred examples of alkoxy include C 1-6 alkoxy (ie, C 1-6 alkyl-O-), C 1-4 alkoxy (ie, C 1-4 alkyl-O-), and the like.
  • C 1-4 alkoxy examples include methoxy (CH 3 O-), ethoxy (CH 3 CH 2 O-), n-propoxy (CH 3 (CH 2 ) 2 O-), isopropoxy ((CH 3 ) 2 CHO-), n-butoxy (CH 3 (CH 2 ) 3 O-), isobutoxy ((CH 3 ) 2 CHCH 2 O-), tert-butoxy ((CH 3 ) 3 CO-), sec-butoxy (CH 3 CH 2 CH(CH 3 )O-) and the like.
  • C 1-6 alkoxy examples include C 1-4 alkoxy, n-pentyloxy (CH 3 (CH 2 ) 4 O-), isopentyloxy ((CH 3 ) 2 CHCH 2 CH 2 O-), Neopentyloxy ((CH 3 ) 3 CCH 2 O-), tert-pentyloxy (CH 3 CH 2 C(CH 3 ) 2 O-), 1,2-dimethylpropoxy (CH 3 CH(CH 3 )CH( Examples include, but are not limited to, CH 3 )O-) and the like.
  • nucleotide and “nucleotide molecule” refer to a nucleoside in which the sugar moiety is a phosphate ester.
  • nucleoside refers to a compound in which a base and a sugar form an N-glycosidic bond. Ribonucleotides whose sugar moiety is D-ribose are obtained by hydrolysis of RNA. Those whose sugar moiety is D-2'-deoxyribose are called deoxyribonucleotides, which are obtained by enzymatic degradation of DNA.
  • nucleotides may be natural or non-natural, or may be artificially synthesized.
  • nucleotide may be modified, and the modification may be made in any one or a combination of the base, sugar moiety, and phosphate moiety, preferably the sugar moiety, the phosphate moiety of the nucleotide, and /or the 3' position of the nucleotide may be modified with a phosphate group.
  • the 3' position of the nucleotide may be modified with a phosphate group, and at least one oxygen atom of the phosphate group may be replaced with a sulfur atom.
  • a representative example of a nucleotide variant may be, but is not limited to, the monomer portion of the compound represented by formula (1).
  • Oligonucleotide and “polynucleotide” are used interchangeably herein and refer to a polymer or oligomer of nucleotides.
  • An “oligonucleotide” may also be referred to as “oligo,” “nucleic acid,” or “nucleic acid strand.”
  • the “oligonucleotide” may be modified, and the modification may be made in any one or a combination of the base, sugar moiety, and phosphate group, and preferably the sugar moiety and the phosphate group of the oligonucleotide may be modified. , and/or the 3' position of the 3' end of the oligonucleotide may be modified with a phosphate group.
  • the 3' position of the 3' end of the oligonucleotide may be modified with a phosphate group, and at least one oxygen atom of the phosphate group may be replaced with a sulfur atom.
  • oligonucleotide variant and “polynucleotide variant” are used interchangeably and refer to a polymer or oligomer of nucleotides and/or nucleotide variants.
  • a typical example of a modified oligonucleotide is a compound represented by formula (1), but is not limited thereto.
  • oligonucleotide variant if the polymer or oligomer contains at least one nucleotide variant, it is referred to as an oligonucleotide variant or a polynucleotide variant.
  • oligonucleotides and oligonucleotide variants are not limited in length as long as two or more nucleotides or variants thereof are polymerized, and unless there is a limitation in other contexts. . Note that this number may be counted as the number of bases in this specification.
  • deoxyribonucleic acid refers to a molecule containing at least one deoxyribonucleotide monomer moiety.
  • ribonucleic acid refers to a molecule that includes at least one ribonucleotide monomer moiety.
  • “Ribonucleotide” means a nucleotide having a hydroxyl group at the 2' position of the ⁇ -D-ribo-furanose moiety.
  • Modification used in the context of nucleic acids refers to the structure of nucleic acids. Refers to a state in which a part or all of the unit or its terminal is substituted with another atomic group, or a functional group is added. A substance (DNA, RNA, nucleic acid, oligonucleotide, etc.) containing such a “modification” is referred to as a "variant”. Modified oligonucleotides include all oligonucleotides that can be alkylated such as methylation, and include any modifications disclosed herein (modifications with various substituents).
  • nucleobase refers to a base component constituting a nucleic acid, including, but not limited to, adenine (A), guanine (G), cytosine (C), Thymine (T), uracil (U), hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, Examples include 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, and 2,6-diaminopurine.
  • terminal monomer portion refers to a portion (typically a nucleoside portion) at the 5' or 3' end of an oligonucleotide.
  • the terminal monomer moiety may be modified, for example, the 2' position may be substituted with a fluoro group.
  • template nucleic acid refers to a nucleic acid having a sequence complementary to an oligonucleotide or a variant thereof.
  • the term "3' end of an oligonucleotide or a variant thereof” refers to the terminal monomer portion bound to the 3' side of the oligonucleotide or a variant thereof.
  • the 5' end of an oligonucleotide or a variant thereof refers to the terminal monomer portion bonded to the 5' side of the oligonucleotide or a variant thereof.
  • linking means being connected via any chemical bond or linker, etc.
  • the chemical bonds include, but are not limited to, covalent bonds, ionic bonds, etc. Including bonds, hydrogen bonds, etc.
  • the linkage of the present disclosure is a covalent bond.
  • condition under which ligation occurs refers to conditions under which oligonucleotides or variants thereof can be ligated.
  • conditions under which ligation occurs are assumed to be conditions in which an enzyme having nucleic acid ligation activity such as ligase is not used. Therefore, conditions for ligation include temperature, pH, solvent, salt concentration (buffer, sodium chloride, etc.), and can be appropriately adjusted by those skilled in the art.
  • continuously complementary sequence and chain length means that the 3' end of the first oligonucleotide or its variant and the 5' end of the second oligonucleotide or its variant are linked. is generated when an oligonucleotide or its variant is complementary to the sequence of a portion of a second oligonucleotide or its variant portion, including a linking portion, from a portion of the first oligonucleotide or its variant portion.
  • the chain length may be long or short as long as it can continuously form a double strand with the first oligonucleotide and the second oligonucleotide. It is preferably 10 or more residues, more preferably 20 to 40 residues.
  • the range that does not prevent the formation of double strands refers to the range in which the oligonucleotide or its variant and the template nucleic acid can form a double strand under the conditions in which the ligation reaction is carried out (reaction temperature, etc.). means.
  • mismatched base pairs within a range that does not prevent the formation of double strands refers to mismatched base pairs that are not complementary sequences that do not allow the oligonucleotide or its variant and template nucleic acid to form a double strand. Refers to mismatched base pairs within the possible range.
  • the term "activator” refers to an agent capable of activating a compound, activating the reaction, and improving the reaction rate and reaction yield.
  • activator for oligonucleotide ligation reaction refers to an activator that can be used when ligating oligonucleotides or variants thereof. Examples include carbodiimide condensing agents, phosphonium condensing agents, uronium condensing agents, and triazine condensing agents.
  • nucleic acid derivative refers to a derivative that is different from a naturally occurring nucleic acid but has a similar function to the original nucleic acid. It also includes chemically modified nucleic acids in the base, sugar, and phosphodiester moieties. Examples include phosphorothioate nucleic acids, LNA-modified nucleic acids, 2'-OMe-modified nucleic acids, 2'-F-modified nucleic acids, 2'-MOE-modified nucleic acids, PMO nucleic acids, PNA nucleic acids, and the like.
  • carbodiimide condensing agent refers to a condensing agent containing carbodiimide in the molecule.
  • EDC also called water soluble carbodiimide (WSC, WSCD)
  • DIPCI diisopropylcarbodiimide
  • DCC dicyclohexylcarbodiimide
  • phosphonium-based condensing agent refers to a condensing agent containing phosphonium in the molecule.
  • PyBroP bromotripyrrolidinophosphonium hexafluorophosphate
  • PyBOP 1H-benzotriazol-1-yloxytris(pyrrolidin-1-yl)phosphonium hexafluorophosphate
  • PyFOP 6-trifluoromethyl-1H- Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate
  • BOP 1-H-benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate
  • PyAOP 7-azabenzotriazole-1 -yloxy tripyrrolidinophosphonium hexafluorophosphate
  • the term "uronium-based condensing agent” refers to a condensing agent containing uronium in the molecule.
  • a condensing agent containing uronium in the molecule O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N , N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) or ⁇ [(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy ⁇ -4-morpholinomethylene ⁇ dimethylammonium hexafluorophosphate (COMU).
  • HATU O-
  • triazine-based condensing agent refers to a condensing agent containing triazine in the molecule.
  • DMT-MM and the like can be mentioned.
  • amidide nucleoside or deoxynucleoside refers to a compound in which the 3' position of a nucleoside or deoxynucleoside has a phosphate ester structure.
  • kit refers to a unit in which parts to be provided are usually divided into two or more compartments. This kit format is preferable when the purpose is to provide a composition that should not be provided mixed for reasons of stability etc., but is preferably mixed immediately before use.
  • raw material refers to a substance used when linking oligonucleotides or variants thereof. Examples include monomers or substituent-introducing reagents, and preferred are amidite or deoxynucleosides, or deoxynucleosides in which the 3' position is a hydroxyl group.
  • the present disclosure provides a method for linking a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof, the method comprising: linking a first oligonucleotide or a variant thereof to a second oligonucleotide or a variant thereof; the step of placing the oligonucleotide or a variant thereof under conditions in which ligation occurs, at the 3' end of the first oligonucleotide or variant thereof and the 5' end of the second oligonucleotide or variant thereof.
  • a method is provided in which ligation occurs and the terminal monomer portion at the 5' end of the second oligonucleotide or variant thereof comprises at least one modification.
  • the technology of the present disclosure by using the technique of linking the first oligonucleotide or its variant and the second oligonucleotide or its variant, the reactivity between the oligonucleotides or their variants can be improved and reaction yields can be achieved. Furthermore, by using the technology of the present disclosure, it is possible to produce long-chain RNA with new functions, and since it is applicable not only to RNA but also to synthetic DNA, functional DNA will also be produced. be able to.
  • the second oligonucleotide or variant thereof used in the present disclosure comprises an optionally substituted amino group, preferably an optionally substituted amino group at the 5' position of its terminal monomer moiety.
  • the amino group included in the second oligonucleotide or variant thereof used in the present disclosure is an unsubstituted amino group.
  • the amino group included in the second oligonucleotide or variant thereof used in the present disclosure is substituted with a C 1-6 alkyl group, illustratively, the amino group used in the present disclosure is substituted with a methyl or ethyl group.
  • the second oligonucleotide or variant thereof used in the present disclosure is substituted with halo or -OH at the 2' position of its terminal monomer moiety, preferably with a halo or -OH at the 2' position of its terminal monomer moiety; The position is replaced by halo. More preferably, the 2' position of the terminal monomer moiety is substituted with a fluoro group.
  • substituting the 2'-position of the terminal monomer moiety it is possible to improve the reactivity between oligonucleotides or their variants and achieve a high reaction yield.
  • the first oligonucleotide used in the present disclosure or a variant thereof is substituted with a hydrogen group or an optionally substituted alkoxy group at the 2' position of the terminal monomer moiety, and preferably, The 2' position of the terminal monomer moiety is substituted with optionally substituted alkoxy. More preferably, the 2' position of the terminal monomer moiety is substituted with methoxy.
  • substituting the 2'-position of the terminal monomer moiety it is possible to improve the reactivity between oligonucleotides or their variants and achieve a high reaction yield.
  • the 2' position of the terminal monomer portion of the first oligonucleotide used in this disclosure or a variant thereof is substituted upward or downward.
  • the 2' position of the terminal monomer portion of the first oligonucleotide or variant thereof is substituted downward.
  • the phosphate group in at least one of the first oligonucleotides or variants thereof used in this disclosure comprises S - or O - .
  • the 3' end of the first oligonucleotide or variant thereof used in the present disclosure includes a phosphate group or a thiophosphate group.
  • the first oligonucleotide or its variant used in the present disclosure and the second oligonucleotide or its variant are linked via a phosphate group or a thiophosphate group.
  • the first oligonucleotide or a variant thereof used in the present disclosure and a plurality of the second oligonucleotides or variants thereof are linked.
  • oligonucleotides or variants thereof having chain lengths of several hundred or 1000 or more can be linked.
  • the conditions for ligation used in this disclosure include using a template nucleic acid.
  • the template nucleic acid has a sequence and length that is continuous and complementary to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof.
  • the first oligonucleotide or its variant has a base sequence of GCUGAAGGGGC from 5' to 3' end
  • the second oligonucleotide or its variant has a base sequence of UUUUGAACUCUGC from 5' to 3' end.
  • the sequence is UCAAAAGCCCUU.
  • the template nucleic acid used in this disclosure is not linked to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof, or to either one of the oligonucleotides or or a variant thereof, or both oligonucleotides or a variant thereof.
  • the template nucleic acid used in this disclosure is not linked to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof.
  • the sequence of the template nucleic acid used in this disclosure is a sequence that is complementary to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof, or does not prevent duplex formation. Contains a range of mismatched base pairs.
  • the conditions for ligation used in the present disclosure are any conditions as long as the ligation proceeds when the provided oligonucleotide or its variant coexists with a template nucleic acid, etc.
  • Those skilled in the art can appropriately determine the presence of other drugs, temperature, pH, solvent, salt concentration (buffer, sodium chloride, etc.), etc.
  • an activator for the ligation reaction of oligonucleotides or variants thereof is used.
  • the activating agent used in the present disclosure is a carbodiimide-based condensing agent, a phosphonium-based condensing agent, a uronium-based condensing agent, or a triazine-based condensing agent, and preferably the carbodiimide-based condensing agent is 1-[3 -(dimethylamino)propyl]-3-ethylcarbodiimide (EDC), diisopropylcarbodiimide (DIPCI), and examples of the phosphonium condensing agent include bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), 1H-benzo Triazol-1-yloxytris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyBOP), 6-trifluoromethyl-1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluor
  • examples of uronium-based condensing agents include O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetra Mention may be made of fluoroborate (TSTU) or ⁇ [(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy ⁇ -4-morpholinomethylene ⁇ dimethylammonium hexafluorophosphate (COMU), and Examples of
  • the activating agent used in the present disclosure is 1-fluoro-2,4-dinitrobenzene (DNFB), or 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC).
  • DNFB 1-fluoro-2,4-dinitrobenzene
  • EDC 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
  • the activators used in the present disclosure include 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt), 1- It is used in combination with hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (HOSu).
  • the first oligonucleotide used in the present disclosure or a variant thereof has the following formula (I):
  • R - is S - or O -
  • Y is a hydrogen group or an alkoxy group
  • B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine) , 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-amino purine, or 2,6-diaminopurine,
  • A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative
  • n is an integer greater than or equal to 1
  • R 1 is a hydroxyl group at the 5'
  • a compound represented by a certain nucleic acid or a nucleic acid derivative A compound represented by a certain nucleic acid or a nucleic acid derivative).
  • the use of such a specific structure makes it possible to create long RNAs with new functions, and can be applied not only to RNA but also to synthetic DNA. Therefore, functional DNA can be produced.
  • by substituting the 2'-position of the terminal monomer moiety it is possible to improve the reactivity between oligonucleotides or their variants and achieve a high reaction yield.
  • Y in the first oligonucleotide or a variant thereof is a methoxy group.
  • n is an integer from 5 to 1000.
  • n is an integer from 5 to 900
  • n is an integer from 5 to 800
  • n is an integer from 5 to 700
  • n is an integer from 5 to 600
  • n is an integer from 5 to 500
  • n is an integer from 5 to 400
  • n is an integer from 5 to 300
  • n is an integer from 5 to 200
  • n is an integer from 5 to 100, and more preferably an integer from 5 to 200.
  • Particularly more preferred is an integer from 10 to 100.
  • the second oligonucleotide used in the present disclosure or a variant thereof has the following formula (II): (wherein, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, p is an integer greater than or equal to 1, and R2 is a nucleic acid whose 3'-position is a hydroxyl group, a phosphate group, or a thiophosphate group, or is a nucleic acid derivative)
  • This is a compound represented by
  • X in the second oligonucleotide or variant thereof is a fluoro group.
  • a fluoro group as X, it is possible to see an improvement in the reactivity between oligonucleotides or their variants and a high reaction yield.
  • p is an integer from 5 to 1000.
  • p is an integer from 5 to 900
  • p is an integer from 5 to 800
  • p is an integer from 5 to 700
  • p is an integer from 5 to 600
  • p is an integer from 5 to 500
  • p is an integer from 5 to 400
  • p is an integer from 5 to 300
  • p is an integer from 5 to 200
  • p is an integer from 5 to 100, and more preferably an integer from 10 to 100.
  • the spacer used in this disclosure is C 1-k alkylene or PEG, preferably optionally substituted C 1-10 alkylene. In some embodiments, the spacer is an optionally substituted C 1-10 alkylene. In some embodiments, the spacer is -(CH 2 ) g -, where g is an integer from 1 to 10. In some embodiments g is 1. In some embodiments g is 2. In some embodiments g is 3. In some embodiments g is 4. In some embodiments g is 5. In some embodiments g is 6. In some embodiments g is 7. In some embodiments g is 8. In some embodiments g is 9. In some embodiments g is 10. Preferably, g is 3 to minimize structural and destabilizing effects on duplex formation.
  • an oligonucleotide or an oligonucleotide thereof in which the 5' end of the first oligonucleotide used in the present disclosure or a variant thereof and the 3' end of the second oligonucleotide used in the present disclosure or a variant thereof are linked.
  • the plurality of variants, the first oligonucleotide or its variant, and the second oligonucleotide or its variant are linked at two or more locations simultaneously or in stages.
  • “simultaneously” refers to linking under one reaction condition
  • stepwise refers to linking not simultaneously but sequentially. When three or more molecules are used, they may be used simultaneously, in stages, or in a combination thereof.
  • the oligonucleotide or variant thereof to which the 5' end of the first oligonucleotide used in the present disclosure or a variant thereof and the template nucleic acid used in the present disclosure are linked is the oligonucleotide or variant thereof used in the present disclosure. linked to an oligonucleotide or a variant thereof.
  • the oligonucleotide or variant thereof used in the present disclosure is linked to the 3' end of the second oligonucleotide or variant thereof used in the present disclosure and the template nucleic acid used in the present disclosure. oligonucleotide or a variant thereof.
  • the linking reaction can be performed without adding a separate template nucleic acid.
  • the 3' end of the first oligonucleotide or variant thereof used in the disclosure and the 5' end of the second oligonucleotide or variant thereof used in the present disclosure are linked.
  • a cyclic oligonucleotide or its variant can be synthesized.
  • a plurality of second oligonucleotides or variants thereof in which the 3'-position of R 2 is a phosphate group or a thiophosphate group are linked in a circle using a plurality of template nucleic acids.
  • the obtained oligonucleotide or its variant becomes circular, and a cyclic oligonucleotide or its variant can be produced.
  • the template nucleic acid used in the present disclosure is an oligonucleotide linked to the 5' end of the first oligonucleotide or variant thereof and the 3' end of the second oligonucleotide or variant thereof used in the present disclosure.
  • the nucleotide or its variant the 5' end of the first oligonucleotide or its variant and the 3' end of the second oligonucleotide or its variant are linked.
  • the present disclosure provides a first oligonucleotide or a variant thereof having a group derived from phosphate at the 3' end and a 5'-amino-5' at the 5' end in the presence of a template nucleic acid.
  • oligonucleotide ligation reaction in which two oligonucleotide fragments are linked by forming a phosphoroamidate bond, 5'-amino-2'-fluoro-2',5, which has a fluoro group introduced at the 2'-position, Oligonucleotide ligation reactions performed using '-dideoxynucleotide monomer moieties are provided.
  • the oligonucleotide monomer portion at the 3'-end of the first oligonucleotide having a group derived from phosphate at the 3'-end is a naturally occurring ribonucleotide, a naturally occurring 2'-deoxynucleotide, Alternatively, it is a 2'-O-methyl nucleotide in which the hydroxyl group at the 2'-position is methylated, or a 2'-substituted-2'-deoxynucleotide derivative in which a substituent such as a fluoro group is introduced at the 2'-position.
  • the template nucleic acid has a first oligonucleotide or variant thereof having a phosphate-derived group at the 3' end and a 5'-amino-5'-deoxynucleotide monomer moiety at the 5' end. Both oligonucleotides of the second oligonucleotide or its variant or its variants have consecutive complementary sequences, or sequences and chain lengths that allow them to form a stable double strand.
  • the template nucleic acid includes not only a nucleic acid independent of the first oligonucleotide or its variant and the second oligonucleotide or its variant, but also a nucleic acid linked to a fragment of either one or both fragments. .
  • each oligonucleotide or variant thereof has a chain length of 5 to 1000 bases. It is preferably 5 to 200 bases, or may be 10 to 100 bases.
  • each of the first oligonucleotide or its variant and the second oligonucleotide or its variant may have an independent sequence, or may be connected into one.
  • the oligonucleotide has a group derived from phosphoric acid at the 3' end and a 5'-amino-5'-deoxynucleotide monomer moiety at the 5' end, and a cyclic oligonucleotide is obtained by a ligation reaction. .
  • the chain length of the template nucleic acid is such that the first oligonucleotide or a variant thereof and the second oligonucleotide or variant thereof to be linked have a sequence that allows for continuous binding, and each oligonucleotide or a variant thereof, including all or a part of the sequence complementary to the variant thereof.
  • the sequence of the template nucleic acid may be a sequence that is completely complementary to each oligonucleotide or a variant thereof, or a sequence that contains mismatched base pairs within a range that does not prevent the formation of double strands.
  • the ligation reaction used in this disclosure has a thiophosphate group at the 3' end of the first oligonucleotide or variant thereof, which is combined with 1-fluoro-2,4-dinitrobenzene (A ligation reaction of oligonucleotides that binds two oligonucleotides or their variants by activation with an activator such as DNFB) and reacting with a second oligonucleotide or its variant to form a phosphoroamidate bond. including.
  • the activator is not limited to 1-fluoro-2,4-dinitrobenzene (DNFB) as long as it is a compound that can activate the phosphoric acid group by reacting with the sulfur atom of thiophosphoric acid.
  • DNFB 1-fluoro-2,4-dinitrobenzene
  • the ligation reaction used in the present disclosure has a phosphate group or thiophosphate group at the 3' end of the first oligonucleotide or variant thereof, which is combined with a carbodiimide-based activator ( (EDC, DCC, etc.) and react with a second oligonucleotide or a variant thereof to form a phosphoroamidate bond, thereby linking the oligonucleotides to link two oligonucleotides or variants thereof.
  • EDC carbodiimide-based activator
  • the ligation reaction used in the present disclosure is performed by treating a first oligonucleotide or a variant thereof having a phosphoric acid-derived group introduced at its 3' end with an activating agent to activate an active intermediate.
  • the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end.
  • an activating agent is added.
  • the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end.
  • a second oligonucleotide having a fluoro-2',5'-deoxynucleotide monomer moiety or a variant thereof, and a template nucleic acid the three oligonucleotides comprising a ligation reaction, all of which are RNA sequences and derivatives thereof.
  • the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end.
  • a ligation reaction in which the second oligonucleotide or variant thereof having a fluoro-2',5'-deoxynucleotide monomer moiety is both an RNA sequence and a derivative thereof, and the template nucleic acid is a DNA sequence and a derivative thereof.
  • the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end.
  • a second oligonucleotide or a variant thereof having a fluoro-2',5'-deoxynucleotide monomer moiety, and a template nucleic acid the three oligonucleotides comprising a ligation reaction, all of which are DNA sequences and derivatives thereof.
  • the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end.
  • a ligation reaction in which the second oligonucleotide or variant thereof having a fluoro-2',5'-deoxynucleotide monomer moiety is both a DNA sequence and a derivative thereof, and the template nucleic acid is an RNA sequence and a derivative thereof.
  • the ligation reaction used in the present disclosure includes a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at the 3' end, 5'-amino-2'-
  • the second oligonucleotide or its variant having a fluoro-2',5'-deoxynucleotide monomer moiety, and all or part of the template nucleic acid sequence consist of an RNA sequence and its derivatives and a DNA sequence and its derivatives.
  • the present disclosure provides a first oligonucleotide or a variant thereof or a second oligonucleotide or a variant thereof, respectively, as described herein.
  • a composition for producing a linked oligonucleotide or a variant thereof using one oligonucleotide or a variant thereof is provided. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
  • composition for producing a linked oligonucleotide or variant thereof using a second oligonucleotide or variant thereof comprising a first oligonucleotide or variant thereof described herein. provide something.
  • composition for producing a linked oligonucleotide or variant thereof using a first oligonucleotide or variant thereof, comprising a second oligonucleotide or variant thereof as described herein. provide something.
  • the composition for producing a linked oligonucleotide or a variant thereof comprises an amidite or deoxynucleoside, or a deoxynucleoside in which the 3' position is a hydroxyl group.
  • the present disclosure provides for producing linked oligonucleotides or variants thereof using template nucleic acids, including a first oligonucleotide or a variant thereof or a second oligonucleotide or a variant thereof, or a combination thereof.
  • template nucleic acids including a first oligonucleotide or a variant thereof or a second oligonucleotide or a variant thereof, or a combination thereof.
  • the present disclosure is a kit for linking oligonucleotides or variants thereof, comprising a first oligonucleotide or variant thereof, a second oligonucleotide or variant thereof, and an activator. and a template nucleic acid.
  • the present disclosure provides a raw material composition for producing a linked oligonucleotide or a variant thereof, including a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
  • the present disclosure provides a raw material composition, wherein the raw material for producing the second oligonucleotide or a variant thereof includes a nucleoside having a fluoro group at the 2' position and an amino group at the 5' position.
  • the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
  • examples of raw materials include nucleosides, monomers, and substituent-introducing reagents.
  • the present disclosure provides a first oligonucleotide or a variant thereof, including a raw material (a monomer or a substituent introduction reagent, etc.) for the first/second oligonucleotide or a variant thereof, and/or a second oligonucleotide or a variant thereof.
  • a raw material a monomer or a substituent introduction reagent, etc.
  • a second oligonucleotide or a variant thereof Compositions for producing oligonucleotides or variants thereof are provided. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
  • the raw material for the first/second oligonucleotide or a variant thereof is an amidite nucleoside or a nucleoside having a hydroxyl group at the 3' position.
  • the present disclosure provides a step of A) obtaining information on a nucleic acid sequence desired to be synthesized; B) synthesizing a plurality of parts of the sequence desired to be synthesized based on the information in A), wherein each the sequence has characteristics of the first and second oligonucleotides or variants thereof, and C) placing them in ligation conditions, optionally in the presence of reagents necessary for the ligation reaction.
  • a method for producing an oligonucleotide having a desired nucleic acid sequence or a variant thereof is provided. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
  • the present disclosure provides a step of A) obtaining information on a nucleic acid sequence desired to be synthesized, B) synthesizing a plurality of parts of the sequence desired to be synthesized based on the information in A), wherein: each sequence has the characteristics of the first and second oligonucleotides or variants thereof; and C) optionally the reagents necessary for the ligation reaction; and B) a nucleic acid comprising the partial sequence synthesized in step B).
  • a method for producing a kit for producing a desired oligonucleotide or a variant thereof which includes the step of packaging the oligonucleotide. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
  • A) the step of obtaining information on the nucleic acid sequence desired to be synthesized includes the step of performing sequence analysis or the like to read the sequence information of the nucleic acid.
  • the reagents necessary for the ligation reaction include a template nucleic acid, an activator. (Novel oligonucleotide or variant thereof)
  • this disclosure provides the following formula: (In the formula, Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine , 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine.
  • X is a hydroxy group or a halogen
  • A is independently at each occurrence a nucleic acid or a nucleic acid derivative
  • n is an integer of 1 or more
  • p is an integer of 1 or more
  • R 1 is a nucleic acid or a nucleic acid derivative in which the 5'-position is a hydroxyl group
  • R 2 is a nucleic acid or a nucleic acid derivative in which the 3'-position is a hydroxyl group, a phosphoric acid group, or a thiophosphoric acid group
  • R 1 and R 2 are may be taken together to form a single bond
  • R 3 is an optionally substituted amino group).
  • Y is a methoxy group
  • each B is independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , said X is a fluoro group, R 1 is hydrogen, and R 2 is a hydroxy group.
  • Y is a methoxy group
  • each B is independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine
  • the X is a fluoro group
  • R 1 and R 2 together form a single bond.
  • Y is a methoxy group.
  • B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1 - methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine, preferably adenine, cytosine, methylcytosine , guanine, thymine, or uracil.
  • B is each independently adenine, cytosine, methylcytosine, guanine, thymine, or uracil.
  • n is an integer from 5 to 1000.
  • n is an integer from 5 to 900
  • n is an integer from 5 to 800
  • n is an integer from 5 to 700
  • n is an integer from 5 to 600
  • n is an integer from 5 to 500
  • n is an integer from 5 to 400
  • n is an integer from 5 to 300
  • n is an integer from 5 to 200
  • n is an integer from 5 to 100, and more preferably an integer from 10 to 100.
  • p is an integer from 5 to 1000.
  • p is an integer from 5 to 900
  • p is an integer from 5 to 800
  • p is an integer from 5 to 700
  • p is an integer from 5 to 600
  • p is an integer from 5 to 500
  • p is an integer from 5 to 400
  • p is an integer from 5 to 300
  • p is an integer from 5 to 200
  • p is an integer from 5 to 100, and more preferably an integer from 10 to 100.
  • X is a hydroxy group or a halogen, preferably a halogen, more preferably a fluoro group.
  • Oligonucleotides in which X is a fluoro group or variants thereof have advantages such as being resistant to nucleases (hard to be degraded) and stabilizing double-strand formation.
  • A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, preferably A is a nucleic acid. More preferably A is DNA or RNA.
  • the nucleic acid derivative is a phosphorothioate nucleic acid, an LNA modified nucleic acid, a 2'-OMe modified nucleic acid, a 2'-F modified nucleic acid, a 2'-MOE modified nucleic acid, a PMO nucleic acid, or a PNA nucleic acid.
  • R 1 is a nucleic acid or a nucleic acid derivative whose 5'-position is a hydroxyl group
  • R 2 is a nucleic acid or a nucleic acid derivative whose 3'-position is a hydroxyl group, a phosphate group, or a thiophosphoric acid group, or R 1 and R 2 may be taken together to form a single bond.
  • R 3 is an optionally substituted amino group.
  • R 3 is an unsubstituted amino group.
  • the amino group of R 3 is substituted by a C 1-6 alkyl group, preferably by a methyl group or an ethyl group.
  • the present disclosure provides a plurality of oligonucleotides or variants thereof in which the 5' end of the first oligonucleotide or variant thereof is linked to the 3' end of the second oligonucleotide or variant thereof;
  • the first oligonucleotide or its variant and the second oligonucleotide or its variant include a linked compound.
  • the present disclosure includes compounds in which R 1 and R 2 are taken together to form a single bond, thereby forming a cyclic oligonucleotide or variant thereof.
  • the present disclosure includes a compound in which a template nucleic acid is attached to the 5' end of a first oligonucleotide or a variant thereof.
  • the present disclosure includes a compound in which a template nucleic acid is attached to the 3' end of a second oligonucleotide or a variant thereof.
  • the present disclosure provides a compound in which the 5' end of a first oligonucleotide or a variant thereof and the 3' end of a second oligonucleotide or variant thereof are linked via a template nucleic acid.
  • compositions and methods of this disclosure can be utilized to treat individuals in need thereof.
  • the individual is a mammal, such as a human, or a non-human mammal.
  • the composition or compound When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, etc. , or injectable organic esters.
  • such pharmaceutical compositions are for administration to humans, particularly by invasive routes (i.e., routes such as injection or implantation that avoid transport or diffusion across epithelial barriers). ), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to effect delayed release of the agent or to selectively target one or more cells, tissues or organs.
  • the pharmaceutical compositions may be prepared in unit dosage forms, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized for reconstitution, powders, solutions, syrups, suppositories, Or it could be an injection.
  • the composition can also be presented in a transdermal delivery system, such as a skin patch.
  • the compositions can also be presented in solutions suitable for topical administration, such as eye drops.
  • the present disclosure can also be widely used in genome editing technology. For example, but not limited to, it can be applied to sgRNA used in genome editing (J. Filippova et al., Biochimie 167 2019 49-60, Ayal Hendel et al. Nature Biotechnology 33 2015 9 85-989 ).
  • oligonucleotides or modified versions thereof (oligonucleic acids) containing the modified (modified) bases of the present disclosure increases activity.
  • One possible application would be to connect fragments of oligonucleic acids containing modified bases synthesized by the method.
  • Another embodiment is to create a relatively long double strand, as described in T. Seya et al. It may also be envisaged to produce it as a template (FIG. 11).
  • long non-coding RNA lncRNA
  • mRNA may also be targeted.
  • Subjects to which administration is contemplated include humans (i.e., men or women of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults). adults (or older adults)) and/or other non-human animals, such as mammals, such as primates (e.g. cynomolgus macaques, rhesus macaques), commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats. , and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys), reptiles, amphibians, and fish.
  • the non-human animal is a mammal.
  • the non-human animal can be male or female at any stage of development.
  • a non-human animal can be a transgenic animal.
  • treat refers to a disease, disorder, or is an act that occurs while suffering from a condition and that reduces the severity of the disease, disorder or condition or delays or slows the progression of the disease, disorder or condition (in the narrow sense of "therapy”);
  • prevention The act of preventing a disease, disorder, or condition from developing or reducing the severity of a disease, disorder, or condition if the subject actually develops the disorder (“prevention”) Included.
  • an "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response, eg, treat a disease, disorder, or condition.
  • a desired biological response eg, treat a disease, disorder, or condition.
  • an effective amount of a compound of the present disclosure will depend on the desired biological endpoint, the pharmacokinetics of the compound, the disease, disorder or condition being treated, the mode of administration, and the age, health, etc. , and may vary depending on factors such as the subject. Effective amounts include therapeutic and prophylactic treatments.
  • a “therapeutically effective amount” of a compound means a “therapeutically effective amount” of a compound that provides a therapeutic effect in the treatment of a disease, disorder or condition or that is associated with the disease, disorder or condition. Refers to an amount sufficient to delay or minimize one or more symptoms.
  • a therapeutically effective amount of a compound means that amount of the therapeutic agent, alone or in combination with other therapies, that provides a therapeutic effect in treating the disease, disorder, or condition.
  • the term "therapeutically effective amount” can include an amount that improves overall therapy, reduces or prevents a symptom or cause of a disease, disorder or condition, or enhances the therapeutic effectiveness of another therapeutic agent.
  • a prophylactically effective amount of a compound means to prevent, prevent, or prevent a disease, disorder, or condition or one or more symptoms associated with the disease, disorder, or condition. or in an amount sufficient to prevent its recurrence.
  • a prophylactically effective amount of a compound means an amount of therapeutic agent that, alone or in combination with other agents, provides a prophylactic effect in preventing the disease, disorder, or condition.
  • the term “prophylactically effective amount” can include an amount that improves overall prophylaxis or enhances the prophylactic effectiveness of another prophylactic agent.
  • Exemplary diseases, disorders or conditions include, but are not limited to, proliferative, neurological, immune, endocrine, cardiovascular, hematological, inflammatory, and disorders characterized by cell death.
  • proliferative diseases, disorders or conditions include cancer, tumors of the hematopoietic system, proliferative breast diseases, proliferative disorders of the lung, proliferative disorders of the colon, proliferative disorders of the liver, and including, but not limited to, ovarian proliferative disorders.
  • oligonucleotides or variants thereof described herein can be used in nucleic acid medicines. (Synthesis example)
  • the compounds of the present disclosure can be produced, for example, by the production methods described below, although they are not limited thereto. These production methods can be modified as appropriate based on the knowledge of those skilled in organic synthetic chemistry. In the following production method, salts of the compounds used as raw materials may be used as long as they do not interfere with the reaction. Paper description (Ueno, Y., Hirai, M., Yoshikawa, K., Kitamura, Y., Hirata, Y., Kiuchi, K., Kitade, Y. Tetrahedron, 2008, 64, 11328-11334., Hideto Maruyama et al., Nucleic Acids Research, 2017, Vol. 45, No.
  • the activated first oligonucleotide or its variant is reacted with a second oligonucleotide or its variant, optionally in the presence of a template nucleic acid, to form the first oligonucleotide or its variant and the second oligonucleotide.
  • Nucleotides or variants thereof can be linked to generate oligonucleotides or variants thereof.
  • Example 1 Synthesis of amidite reagent of 5'-amino-5'-deoxynucleoside Silicagel 70 TLC Plate (Fuji Film Wako Pure Chemical Industries, Ltd.) was used for thin layer chromatography. Wakogel C-200 (Fuji Film Wako Pure Chemical Industries) and Silica Gel 60 (Nacalai Tesque) were used for column chromatography. 1 H NMR and 13 C NMR were measured using a Bruker Avance III 500 MHz using tetramethylsilane as an internal standard. Mass spectrometry was measured using Thermo Scientific Exactive installed at Hokkaido University Global Facility Center.
  • a 1N sodium hydroxide solution (2.7 mL) was added to the reaction solution, stirred at room temperature for 1 hour, and then concentrated under reduced pressure. After adding water (75 mL) and a saturated aqueous sodium hydrogen carbonate solution (75 mL) to the reaction solution, the mixture was extracted three times with ethyl acetate (60 mL). Furthermore, the organic layer was washed once with saturated brine (60 mL) and dried over sodium sulfate. The solution was concentrated under reduced pressure to obtain a white solid material.
  • Tetrahedron, 2002, 58, 867-879. Maruyama, H., Oikawa , R., Hayakawa, M., Takamori, S., Kimura, Y., Abe, N., Tsuji, G., Matsuda, A., Shuto, S., Ito, Y., Abe, H. Nucleic Acids Res., 2017, 45, 7042-7048.).
  • the synthesized oligonucleotides were purified by high performance liquid chromatography (HPLC). HPLC was performed by connecting a Waters ⁇ -Bondasphere C18 300A (inner diameter 3.9 mm x length 150 mm, Waters) to a Gilson device. As mobile phase, a concentration gradient of acetonitrile in 0.1 M triethylammonium acetate buffer (TEAA, pH 7.0) was used in the case of reversed phase. The types of oligonucleotides synthesized and the conditions for reverse phase HPLC are shown below. Solution A: 5% acetonitrile/0.1M TEAA (pH 7.0) Solution B: 25% acetonitrile/0.1M TEAA (pH 7.0)
  • Example 2 (Preparation of modified DNFB (2Fps-DNFB) (Example 2)) A 2 Fps DNFB modified product (Example 2) was produced by the following method. Nucleic acid strand 2Fps ( Figure 3) (1.0 nmol) containing 3'-phosphorothioate modification was mixed with 10 mM 1-fluoro-2,4-dinitrobenzene (DNFB), 10% dimethyl sulfoxide (DMSO), and 20 mM sodium borate (pH 8). .5). After reaction at room temperature for 2 hours, crude purification was performed using a gel filtration column (GE Healthcare, NAP-5).
  • DNFB 1-fluoro-2,4-dinitrobenzene
  • DMSO dimethyl sulfoxide
  • pH 8 sodium borate
  • the DNFB-modified product of 2Meps was prepared by using the nucleic acid chain 2Meps (FIG. 3) containing 3'-phosphorothioate modification and performing a reaction in the same manner as the above-mentioned 2Fps-DNFB (Example 2).
  • RNA conjugate (2Me-2OH5N) (Example 3)
  • the conjugate 2Me-2OH5N (10-13) was produced by the following method. Nucleic acid chain 2OH5N ( Figure 3) (2.5 pmol) containing 5' amino modification and modified with a fluorescent molecule and template nucleic acid chain tmr27 ( Figure 3) (5 pmol) were mixed in 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8). ) (total volume: 12.5 ⁇ L). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1).
  • Nucleic acid chain 2Meps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 ⁇ L) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 ⁇ L. After reacting for 30, 60, and 120 minutes, 5 ⁇ L was mixed with 5 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate.
  • loading buffer 80% formamide, 10 mM EDTA
  • RNA conjugate (2Me-2F5N) (Example 4)
  • the conjugate 2Me-2F5N(10-13) (Example 4, FIG. 3) was produced by the following method.
  • a nucleic acid chain 2OH5N ( Figure 3) (2.5 pmol) containing a 5' amino modification and modified with a fluorescent molecule and a template nucleic acid chain tmr27 ( Figure 3) (5 pmol) were mixed in 20 mM sodium phosphate, 10 mM magnesium chloride ( pH 8) (total volume: 12.5 ⁇ L).
  • the reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1).
  • Nucleic acid chain 2Meps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 ⁇ L) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 ⁇ L. After reacting for 30, 60, and 120 minutes, 5 ⁇ L was mixed with 5 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate.
  • loading buffer 80% formamide, 10 mM EDTA
  • Example 4 Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 4). . More specifically, as shown in FIG. 6A, it was confirmed that the conjugate of Example 4 migrated later than the nucleic acid strand of the fluorescent molecule-modified nucleic acid strand 2F5N, which was the raw material.
  • RNA conjugate (2F-2OH5N) (Example 5)
  • the conjugate 2F-2OH5N was produced by the following method.
  • a nucleic acid chain 2OH5N ( Figure 3) (2.5 pmol) containing a 5' amino modification and modified with a fluorescent molecule and a template nucleic acid chain tmr27 ( Figure 3) (5 pmol) were mixed in 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8). ) (total volume: 12.5 ⁇ L).
  • the reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1).
  • Nucleic acid chain 2Fps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 ⁇ L) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 ⁇ L. After reacting for 30, 60, and 120 minutes, 5 ⁇ L was mixed with 5 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate.
  • loading buffer 80% formamide, 10 mM EDTA
  • Example 5 Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6 M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 5). . More specifically, as shown in FIG. 6A, it was confirmed that the conjugate of Example 5 migrated later than the 2OH5N nucleic acid strand modified with a fluorescent molecule as a raw material.
  • Nucleic acid chain 2Fps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 ⁇ L) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 ⁇ L. After reacting for 30, 60, and 120 minutes, 5 ⁇ L was mixed with 5 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate.
  • loading buffer 80% formamide, 10 mM EDTA
  • Example 3 Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). . More specifically, as shown in FIG. 6A, it was confirmed that the conjugate of Example 6 migrated later than the 2F5N nucleic acid strand modified with a fluorescent molecule as a raw material. Furthermore, as shown in Figure 6B, the amount of conjugates observed increased with the passage of reaction time, and the reaction yield was higher than that of the above 2F-2OH5N at all times, so 2F5N was modified with DNFB. It was confirmed that it is more effective than 2OH5N in ligation reactions with nucleic acids.
  • RNA conjugate (Example 7) using DNA template)
  • 2Me-2OH5N, 2M-2F5N, 2F-2OH5N, and 2F-2F5N were similarly produced by adding DNA template tmd27 instead of RNA template tmr27.
  • 2M-2F5N showed a higher reaction yield compared to 2Me-2OH5N
  • 2F-2F5N showed a higher reaction yield compared to 2F-2OH5N. It was confirmed that 2F5N is more effective than 2OH5N in the ligation reaction with DNFB-modified nucleic acids.
  • RNA conjugate (2Me-2OH5N) (Example 8) using activator (DNFB)
  • DNFB activator
  • a conjugate of 2Me-2OH5N (Example 8, FIG. 3) was produced by the following method. Nucleic acid strand 2Meps containing 3'-phosphorothioate modification (Figure 3) (50 pmol), nucleic acid strand 2OH5N containing 5' amino modification and modified with a fluorescent molecule ( Figure 3) (10 pmol), and template nucleic acid strand tmr27 (20 pmol) was dissolved in a solution (total volume 18 ⁇ L) containing 50 mM sodium phosphate and 100 mM sodium chloride (pH 8).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Add 2 ⁇ L of DMSO solution containing 100 mM DNFB to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 6 and 24 hours, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to prepare the conjugate. A solution containing was obtained. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). . More specifically, as shown in FIG. 9A, the amount of conjugate reacted increased as the reaction time progressed.
  • RNA conjugate (2Me-2F5N) (Example 9) using activator (DNFB)
  • a conjugate of 2Me-2F5N (Example 9, FIG. 3) was produced by the following method using DNFB.
  • Nucleic acid strand 2Meps containing 3'-phosphorothioate modification (Fig. 3) (50 pmol)
  • template nucleic acid strand tmr27 (20 pmol) was dissolved in a solution (total volume 18 ⁇ L) containing 50 mM sodium phosphate and 100 mM sodium chloride (pH 8).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Add 2 ⁇ L of DMSO solution containing 100 mM DNFB to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 6 and 24 hours, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to prepare the conjugate. A solution containing was obtained. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). . More specifically, as shown in FIG. 9A, the amount of conjugate reacted increased as the reaction time progressed.
  • RNA conjugate (Example 10) using DNA template and activator (DNFB)
  • DNFB DNA template and activator
  • the above 2Me-2OH5N and 2Me-2F5N were similarly produced using DNA template tmd27.
  • Figure 9B similar to the RNA template, 2Me-2F5N showed a higher reaction yield compared to 2Me-2OH5N, and 2F-2F5N showed a higher reaction yield compared to 2F-2OH5N.
  • 2F5N was confirmed to be more effective than 2OH5N in ligation reactions using DNFB.
  • reaction Solution 3 (total volume 18 ⁇ L) containing 50 mM sodium phosphate and 100 mM sodium chloride (pH 8). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Add 2 ⁇ L of DMSO solution containing 100 mM DNFB to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 6 hours, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to prepare a solution containing the conjugate. I got it.
  • loading buffer 80% formamide, 10 mM EDTA
  • RNA conjugate (Example 13) using DNA template and activator (DNFB)
  • DNFB DNA template and activator
  • the above 2Me-2OH5N_C and 2Me-2F5N_C were similarly produced using DNA template tmd27-II.
  • 2Me-2F5N_C showed a higher reaction yield compared to 2Me-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction using DNFB. did.
  • Tm measurement of 5'oligo and template DNA and RNA Melting curves with the template nucleic acid were measured for nucleic acid strands 2Meps and 2Fps (FIG. 3) containing 3'-phosphorothioate modification.
  • DNA tmr27 (FIG. 3) and RNA tmd27 (FIG. 3) were selected as target nucleic acids.
  • the nucleic acid strand 2Fps containing the 3'-phosphorothioate modification showed a higher Tm value than 2Meps in both the RNA template and the DNA template. Furthermore, 2F5N containing 5' amino modification showed a higher Tm value than 2OH5N in both RNA template and DNA template. Table 1. Tm analysis of 5'-oligo or 3'-oligo with template nucleic acid
  • RNA conjugate (2Me-2OH5N) (Example 14) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • the conjugate 2Me-2OH5N (Example 14, FIG. 3) was produced by the following method. Nucleic acid strand 2Mepo (Fig. 3) (20 pmol), nucleic acid strand 2OH5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 ⁇ L) containing HEPES and 1M sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 60 minutes, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging.
  • 6M EDC Add 2 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 60 minutes, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate
  • Example 14 migrated later than the 2OH5N nucleic acid chain modified with a fluorescent molecule as a raw material.
  • Gel analysis was performed using Typhoon FLA9000 (GE Health.) after electrophoresis.
  • RNA conjugate (2Me-2F5N) (Example 15) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • a conjugate of 2Me-2F5N (Example 15, FIG. 3) was produced by the following method using EDC.
  • Nucleic acid strand 2Mepo (Fig. 3) (20 pmol)
  • nucleic acid strand 2F5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule
  • template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 ⁇ L) containing HEPES and 1M sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 60 minutes, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • 2Me-2F5N showed a higher reaction yield compared to 2Me-2OH5N, confirming that 2F5N is more effective than 2OH5N in the ligation reaction with the nucleic acid chain 2Mepo using carbodiimide. did.
  • RNA conjugate (2F-2OH5N) (Example 16) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • a conjugate of 2F-2OH5N (Example 16, FIG. 3) was prepared using EDC in the following manner. Nucleic acid strand 2Fpo (Fig. 3) (20 pmol), nucleic acid strand 2OH5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 ⁇ L) containing HEPES and 1M sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 60 minutes, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • RNA conjugate (2F-2F5N) (Example 17) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • the conjugate 2F-2F5N (Example 17, FIG. 3) was produced by the following method. Nucleic acid strand 2Fpo (Fig. 3) (20 pmol), nucleic acid strand 2F5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 ⁇ L) containing HEPES and 1M sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 60 minutes, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • 2F-2F5N showed a higher reaction yield compared to 2F-2OH5N, confirming that 2F5N is more effective than 2OH5N in the ligation reaction with the nucleic acid chain 2Fpo using carbodiimide. did.
  • RNA conjugate (2H-2OH5N) (Example 18) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • the conjugate 2H-2OH5N (Example 18, FIG. 3) was produced by the following method. Nucleic acid strand 2Hpo (Fig. 3) (20 pmol), nucleic acid strand 2OH5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 ⁇ L) containing HEPES and 1M sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 60 minutes, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • RNA conjugate (2H-2F5N) (Example 19) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • the conjugate 2H-2F5N (Example 19, FIG. 3) was produced by the following method. Nucleic acid strand 2Hpo (Fig. 3) (20 pmol), nucleic acid strand 2F5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 ⁇ L) containing HEPES and 1M sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 20 ⁇ L at 27°C for 60 minutes, and then mix 3 ⁇ L with 12 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • RNA conjugate (2Me-2OH5N) (Example 20) using carbodiimide-based activator (EDC)
  • EDC carbodiimide-based activator
  • Nucleic acid strand 2Mepo (Fig. 3) (500 pmol)
  • nucleic acid strand 2OH5N containing 5' amino modification (Fig. 3)
  • template nucleic acid strand tmr12 (Fig. 3) (500 pmol) were mixed in 250 mM HEPES, 1 M sodium chloride ( pH 7.3) (total volume: 90 ⁇ L).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. A solution containing HCl was added to 10 ⁇ L of reaction solution 1, and the reaction was carried out at 17° C. for 6 hours in a total volume of 100 ⁇ L, followed by rough purification using a gel filtration column (GE Healthcare, NAP-5). Thereafter, it was purified using HPLC using a reverse phase column to obtain 2Me-2OH5N. The following solution was used for purification.
  • Solution A 5% acetonitrile/0.1M TEAA (pH 7.0)
  • Solution B 25% acetonitrile/0.1M TEAA (pH 7.0) More specifically, as shown in FIG. 10V, it was confirmed that the peak of the RNA linkage 2Me-2OH5N of Example 20 was observed later than that of the raw materials 2Mepo and 2OH5N.
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 30 ⁇ L at 37°C for 30 minutes, and then mix 5 ⁇ L with 15 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging.
  • loading buffer 80% formamide, 10 mM EDTA
  • Example 22 migrated later than the nucleic acid strand of the fluorescent molecule-modified nucleic acid strand 2OH5N_C, which was the raw material.
  • Gel analysis was performed using Typhoon FLA9000 (GE Health.) after electrophoresis.
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 30 ⁇ L at 37°C for 30 minutes, and then mix 5 ⁇ L with 15 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • 2Me-2F5N_C showed a higher reaction yield compared to 2Me-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction with the nucleic acid chain 2Mepo using carbodiimide. did.
  • RNA conjugate (2F-2OH5N_C) (Example 24) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • a conjugate, 2F-2OH5N_C (Example 24, FIG. 3), was produced using EDC in the following manner.
  • Nucleic acid strand 2Fpo (Fig. 3) (3 pmol)
  • nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Fig. 3) (3 pmol)
  • template nucleic acid strand tmd27-II (Fig. 3) (3 pmol). was dissolved in a solution (total volume 27 ⁇ L) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 30 ⁇ L at 37°C for 30 minutes, and then mix 5 ⁇ L with 15 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 30 ⁇ L at 37°C for 30 minutes, and then mix 5 ⁇ L with 15 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • 2F-2F5N_C showed a higher reaction yield compared to 2F-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction with the nucleic acid chain 2Fpo using carbodiimide. did.
  • RNA conjugate (2H-2OH5N_C) (Example 26) using carbodiimide-based activator (EDC))
  • EDC carbodiimide-based activator
  • the conjugate 2H-2OH5N_C (Example 26, FIG. 3) was produced by the following method. Nucleic acid strand 2Hpo ( Figure 3) (3 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule ( Figure 3) (3 pmol), and template nucleic acid strand tmd27-II ( Figure 3) (3 pmol). was dissolved in a solution (total volume 27 ⁇ L) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 30 ⁇ L at 37°C for 30 minutes, and then mix 5 ⁇ L with 15 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • reaction Solution 1 The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 ⁇ L of a solution containing HCl to reaction solution 1, react in a total volume of 30 ⁇ L at 37°C for 30 minutes, and then mix 5 ⁇ L with 15 ⁇ L of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
  • 2H-2F5N_C showed a higher reaction yield compared to 2H-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction with nucleic acid chain 2Hpo using carbodiimide. did.
  • Example 28 The 5' end of the oligonucleotide or its variant (the first oligonucleotide or its variant) whose 3' position is modified with a phosphate group, and the oligonucleotide or its variant whose 5' position is amino-modified (the first oligonucleotide or its variant).
  • a nucleotide or a variant thereof) is reacted with an oligonucleotide or a variant thereof that is amino-modified at the 5' position (a second oligonucleotide or a variant thereof) in the presence of an activator.
  • the above is linked simultaneously or stepwise to produce an oligonucleotide or a variant thereof in which a plurality of oligonucleotides or variants thereof are linked.
  • Example 29 An oligonucleotide or a variant thereof in which the template nucleic acid is amino-modified at the 5' position or a variant thereof (a second oligonucleotide or a variant thereof), and an oligonucleotide or a variant thereof that is amino-modified at the 5' position.
  • a phosphate group-modified oligonucleotide or a variant thereof is reacted in the presence of an activator to produce a linked oligonucleotide or a variant thereof.
  • Example 30 An oligonucleotide or a variant thereof in which the template nucleic acid is linked to the 5' end of an oligonucleotide or a variant thereof (first oligonucleotide or a variant thereof) whose 3' position is modified with a phosphate group;
  • a linked oligonucleotide or a variant thereof is produced by reacting an oligonucleotide amino-modified at the ' position or a variant thereof (a second oligonucleotide or a variant thereof) in the presence of an activating agent. do.
  • Both ends of the template nucleic acid are linked to the 5' end of an oligonucleotide or a variant thereof (the first oligonucleotide or a variant thereof) whose 3' position is modified with a phosphate group, and the 5' position is amino-modified.
  • the oligonucleotide or its variant linked to the 3' end of the oligonucleotide or its variant (second oligonucleotide or its variant) is reacted in the presence of an activator to form the oligonucleotide or its variant.
  • a cyclic oligonucleotide or a variant thereof in which the 3' end and 5' end of the variant are linked is produced.
  • Example 32 Formulation example
  • Compounds of the present disclosure may be provided in any suitable dosage form.
  • An example is shown below. Examples include tablets, capsules, powders, granules, solutions, suspensions, injections, patches, poultices, and the like. Further, these preparations can be manufactured by a known method using additives commonly used as pharmaceutical additives, including appropriately mixing them.
  • Example 33 Animal experiment
  • a diseased mouse eg, a tumor-bearing mouse
  • the oligonucleotide of the present disclosure or a placebo is administered intraperitoneally.
  • a comparison between a placebo and the oligonucleotide can be performed to evaluate the effectiveness of the oligonucleotide of the present disclosure against diseases in the mouse body.
  • Example 34 Create a relatively long double strand as described in T. Seya et al. Advanced Drug Delivery Reviews 147 2019 37-43 without using template DNA so that each strand serves as a template It can also be assumed (FIG. 11).
  • a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof are reacted in the presence of an activator to form a linked oligonucleotide or its variant. Create a variant.
  • the technology provided in the present disclosure can be used in any field that uses technology for linking oligonucleotides or variants thereof.
  • RNA template (tmr27) Sequence number 2: DNA template (tmd27) Sequence number 3: RNA template (tmr12) SEQ ID NO: 4: First oligonucleotide: 5'oligo (cytidine) SEQ ID NO: 5: First oligonucleotide: 5'oligo (2'O-methyl-cytidine) SEQ ID NO: 6: Second oligonucleotide: 3'oligo (2'H-Uridine) SEQ ID NO: 7: First oligonucleotide + template nucleic acid, tmr12+5'oligo (cytidine) SEQ ID NO: 8: Template nucleic acid + first oligonucleotide, tmr12 + 5'oligo (2'O-methyl-cytidine) SEQ ID NO: 9: Second oligonucleotide + template nucleic acid, 3'oligo (2'H-Uridine) + tmr12 SEQ ID NO: 5

Abstract

Provided is a new technology for a chemical ligation reaction of oligonucleotides. When 2'-fluoro-5'-amino-nucleic acid (2F-5N nucleic acid) having a fluorine atom at the 2'-positon of 5'-amino-nucleic acid (5N nucleic acid) is synthesized, and an oligonucleotide having the same at a terminal or a modified product thereof is used in ligation, it is possible to expect a stable bond with a template nucleic acid by means of a 2'-fluoro modification in the 2F-5N nucleic acid and an improved reactivity of the 5'-amino group. In addition, the fluorine atom, because of having a hydrogen bond similarly to a 2'-hydroxyl group of a nucleic acid, is suitable for interaction with a protein, and enables obtaining of a synergistic effect of combining with an amino group in a ligation reaction because of having resistance against nucleases.

Description

オリゴヌクレオチドの化学的連結反応Chemical ligation of oligonucleotides
　本開示は、長鎖のオリゴヌクレオチドまたはポリヌクレオチドまたはその改変体の合成のための化学的連結反応およびその応用に関する。 The present disclosure relates to chemical ligation reactions for the synthesis of long oligonucleotides or polynucleotides or variants thereof and their applications.
　長鎖のＲＮＡ分子は化学合成することは困難なため、ＲＮＡ合成酵素を用いた転写反応によって作製される場合が多い。しかしながら酵素を用いる転写反応では化学修飾した核酸を部位特異的に導入することは困難である。修飾核酸は、生体内における核酸分解酵素に対する耐性の増強の他、タンパク質と高い親和性を発揮させ、蛍光分子などによる標識など新たな機能をＲＮＡに付与することには不可欠である。 Since long-chain RNA molecules are difficult to chemically synthesize, they are often produced by a transcription reaction using RNA synthase. However, in transcription reactions using enzymes, it is difficult to site-specifically introduce chemically modified nucleic acids. Modified nucleic acids are essential for increasing resistance to nucleolytic enzymes in vivo, exhibiting high affinity with proteins, and imparting new functions to RNA, such as labeling with fluorescent molecules.
　本開示は、オリゴヌクレオチドまたはポリヌクレオチド（本明細書において、代表して「オリゴヌクレオチド」といい、オリゴヌクレオチドという場合は長さに特に限定はないことが理解される。）またはその改変体の新たな連結方法に関する。限定を意図しない一つの実施形態では、５’－アミノ－核酸（５Ｎ核酸）の２’位にフッ素原子を有する２’－フルオロ－５’－アミノ－核酸（２Ｆ－５Ｎ核酸）を合成し、それを末端に有するオリゴヌクレオチドまたはポリヌクレオチドまたはその改変体を連結に用いることで、２Ｆ－５Ｎ核酸の２’－フルオロ修飾による、テンプレート核酸との安定な結合と５’－アミノ基の反応性の向上が期待できる。加えて、フッ素原子は核酸の２’水酸基と同じく水素結合を有するため、タンパク質との相互作用にも適しており、核酸分解酵素に対する耐性も高いことから連結反応におけるアミノ基とあわせる相乗効果が得られる。 The present disclosure relates to new oligonucleotides or polynucleotides (herein, typically referred to as "oligonucleotides"; when referred to as oligonucleotides, it is understood that there is no particular limitation in length) or variants thereof. Concerning connection methods. In one non-limiting embodiment, a 2'-fluoro-5'-amino-nucleic acid (2F-5N nucleic acid) having a fluorine atom at the 2' position of a 5'-amino-nucleic acid (5N nucleic acid) is synthesized, By using oligonucleotides or polynucleotides having this terminus or their variants for linkage, the 2F-5N nucleic acid can be stably bound to the template nucleic acid by 2'-fluoro modification, and the reactivity of the 5'-amino group can be improved. We can expect improvement. In addition, since fluorine atoms have hydrogen bonds like the 2' hydroxyl groups of nucleic acids, they are suitable for interaction with proteins, and have high resistance to nucleolytic enzymes, so they can have a synergistic effect when combined with amino groups in ligation reactions. It will be done.
　本開示により、限定を意図しないが、化学修飾したＲＮＡ分子を化学的に高効率で結合させることができ、新たな機能を有した長鎖ＲＮＡの作製が可能になる（その代表図として、図１）。またこの反応は、ＲＮＡのみならず合成ＤＮＡに対しても適用可能なことから、機能性ＤＮＡの作製にも有効な技術が提供される。
　本発明は、例えば、以下の項目を提供する。
(項目１)
　第一のオリゴヌクレオチドまたはその改変体と第二のオリゴヌクレオチドまたはその改変体とを連結させる方法であって、
　該第一のオリゴヌクレオチドまたはその改変体と該第二のオリゴヌクレオチドまたはその改変体とを、連結が生じる条件下に配置する工程を含み、
　該第一のオリゴヌクレオチドまたはその改変体の３’末端と、該第二のオリゴヌクレオチドまたはその改変体の５’末端において連結が生じ、第二のオリゴヌクレオチドの５’末端側の末端モノマー部分が少なくとも一つの改変を含む、
方法。
(項目２)
　前記第一のオリゴヌクレオチドまたはその改変体の３’末端は、リン酸基またはチオリン酸基を含む、先行する項目のいずれか一項に記載の方法。
(項目３)
　前記第二のオリゴヌクレオチドまたはその改変体は、置換されていてもよいアミノ基を含む、先行する項目のいずれか一項に記載の方法。
(項目４)
　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の５’位に置換されていてもよいアミノ基を含む、先行する項目のいずれか一項に記載の方法。
(項目５)
　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の５’位に置換されていないアミノ基を含む、先行する項目のいずれか一項に記載の方法。
(項目６)
　前記アミノ基が、Ｃ１－６アルキル基によって置換されている、先行する項目のいずれか一項に記載の方法。
(項目７)
　前記アミノ基がメチル基またはエチル基によって置換されている、先行する項目のいずれか一項に記載の方法。
(項目８)
　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がハロまたは－ＯＨで置換されている、先行する項目のいずれか一項に記載の方法。
(項目９)
　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がハロで置換されている、先行する項目のいずれか一項に記載の方法。
(項目１０)
　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がフルオロ基で置換されている、先行する項目のいずれか一項に記載の方法。
(項目１１)
　前記第一のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位が水素基または置換されていてもよいアルコキシ基で置換されている、先行する項目のいずれか一項に記載の方法。
(項目１２)
　前記第一のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位が置換されていてもよいアルコキシで置換されている、先行する項目のいずれか一項に記載の方法。
(項目１３)
　前記第一のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がメトキシで置換されている、先行する項目のいずれか一項に記載の方法。
(項目１４)
　前記末端モノマー部分の２’位が上向きで置換されている、先行する項目のいずれか一項に記載の方法。
(項目１５)
　前記末端モノマー部分の２’位が下向きで置換されている、先行する項目のいずれか一項に記載の方法。
(項目１６)
　前記第一のオリゴヌクレオチドまたはその改変体と前記第二のオリゴヌクレオチドまたはその改変体が、リン酸基またはチオリン酸基を介して連結している、先行する項目のいずれか一項に記載の方法。
(項目１７)
　前記連結が生じる条件は、テンプレート核酸を用いることを包含する、先行する項目のいずれか一項に記載の方法。
(項目１８)
　前記テンプレート核酸が、前記第一のオリゴヌクレオチドまたはその改変体および前記第二のオリゴヌクレオチドまたはその改変体と連続して相補的な配列および鎖長を有する、先行する項目のいずれか一項に記載の方法。
(項目１９)
　前記テンプレート核酸の鎖長が、１０残基以上である、先行する項目のいずれか一項に記載の方法。
(項目２０)
　前記テンプレート核酸の鎖長が、２０～４０残基である、先行する項目のいずれか一項に記載の方法。
(項目２１)
　前記テンプレート核酸が、前記第一のオリゴヌクレオチドもしくはその改変体および前記第二のオリゴヌクレオチドもしくはその改変体とは連結していないか、または、いずれか一方のオリゴヌクレオチドもしくはその改変体、もしくは両方のオリゴヌクレオチドもしくはその改変体と連結している、先行する項目のいずれか一項に記載の方法。
(項目２２)
　前記テンプレート核酸の配列は、前記第一のオリゴヌクレオチドまたはその改変体および前記第二のオリゴヌクレオチドまたはその改変体と相補的な配列、あるいは二本鎖の形成を妨げない範囲のミスマッチ塩基対を含む、先行する項目のいずれか一項に記載の方法。
(項目２３)
　前記連結が生じる条件は、オリゴヌクレオチド連結反応の活性化剤の使用を包含する、先行する項目のいずれか一項に記載の方法。
(項目２４)
　前記第一のオリゴヌクレオチドまたはその改変体が、下記式（Ｉ）

（式中、Ｒ－はＳ－またはＯ－であり、Ｙは水素基またはアルコキシ基であり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｎは１以上の整数であり、Ｒ１は５’－位が水酸基である核酸、または核酸誘導体である）
で示される化合物である、先行する項目のいずれか一項に記載の方法。
(項目２５)
　前記Ｙがメトキシ基である、先行する項目のいずれか一項に記載の方法。
(項目２６)
　ｎが５から１０００の整数である、先行する項目のいずれか一項に記載の方法。
(項目２７)
　前記第二のオリゴヌクレオチドまたはその改変体が、下記式（ＩＩ）

（式中、Ｘは、ヒドロキシ基またはハロゲンであり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｐは１以上の整数であり、Ｒ２は３’－位が水酸基、リン酸基またはチオリン酸基である核酸、または核酸誘導体である）
で示される化合物である、先行する項目のいずれか一項に記載の方法。
(項目２８)
　前記Ｘがフルオロ基である、先行する項目のいずれか一項に記載の方法。
(項目２９)
　ｐが５から１０００の整数である、先行する項目のいずれか一項に記載の方法。
(項目３０)
　前記スペーサーが置換されていてもよいＣ１－１０アルキレンまたはＰＥＧである、先行する項目のいずれか一項に記載の方法。
(項目３１)
　前記スペーサーがＣ３アルキレンである、先行する項目のいずれか一項に記載の方法。
(項目３２)
　前記活性化剤が、カルボジイミド系縮合剤、ホスホニウム系縮合剤、ウロニウム系縮合剤またはトリアジン系縮合剤である、先行する項目のいずれか一項に記載の方法。
(項目３３)
　前記カルボジイミド系縮合剤が１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）、ジイソプロピルカルボジイミド（ＤＩＰＣＩ）またはジシクロヘキシルカルボジイミド（ＤＣＣ）である、先行する項目のいずれか一項に記載の方法。
(項目３４)
　前記ホスホニウム系縮合剤がブロモトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＢｒｏＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ピロリジン－１－イル）ホスホニウム・ヘキサフルオロホスファート（ＰｙＢＯＰ）、６－トリフルオロメチル－１Ｈ－ベンゾトリアゾール－１－イルオキシトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＦＯＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ジメチルアミノ）　ホスホニウムヘキサフルオロホスファート（ＢＯＰ）または（７－アザベンゾトリアゾール－１－イルオキシ）トリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＡＯＰ）である、先行する項目のいずれか一項に記載の方法。
(項目３５)
　前記ウロニウム系縮合剤がＯ－（７－アザベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（ＨＡＴＵ）、Ｏ－（ベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（ＨＢＴＵ）、Ｏ－（Ｎ－スクシンイミジル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウム　テトラフルオロほう酸塩（ＴＳＴＵ）または｛｛［（１－シアノ－２－エトキシ－２－オキソエチリデン）アミノ］オキシ｝－４－モルホリノメチレン｝ジメチルアンモニウム　ヘキサフルオロホスファート（ＣＯＭＵ）である、先行する項目のいずれか一項に記載の方法。
(項目３６)
　前記トリアジン系縮合剤がＤＭＴ－ＭＭである、先行する項目のいずれか一項に記載の方法。
(項目３７)
　前記活性化剤が、１－フルオロ－２，４－ジニトロベンゼン（ＤＮＦＢ）、または１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）である、先行する項目のいずれか一項に記載の方法。
(項目３８)
　前記活性化剤が、１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）と１－ヒドロキシ－７－アザベンゾトリアゾール（ＨＯＡｔ）、１－ヒドロキシベンゾトリアゾール（ＨＯＢｔ）またはＮ－ヒドロキシコハク酸イミド（ＨＯＳｕ）とを組み合わせて使用される、先行する項目のいずれか一項に記載の方法。
(項目３９)
　前記第一のオリゴヌクレオチドまたはその改変体の５’末端と前記第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体の複数と、前記第一のオリゴヌクレオチドまたはその改変体と、前記第二のオリゴヌクレオチドまたはその改変体とが、2か所以上同時にまたは段階的に連結される、先行する項目のいずれか一項に記載の方法。
(項目４０)
　前記第一のオリゴヌクレオチドまたはその改変体の５’末端および前記テンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、前記第二のオリゴヌクレオチドまたはその改変体と連結される、先行する項目のいずれか一項に記載の方法。
(項目４１)
　前記第二のオリゴヌクレオチドまたはその改変体の３’末端および前記テンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、前記第一のオリゴヌクレオチドまたはその改変体と連結される、先行する項目のいずれか一項に記載の方法。
(項目４２)
　前記第一のオリゴヌクレオチドまたはその改変体の５’末端と第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体と、前記第一のオリゴヌクレオチドまたはその改変体の３’末端と、第二のオリゴヌクレオチドまたはその改変体の５’末端とが、連結される、先行する項目のいずれか一項に記載の方法。
(項目４３)
　Ｒ２の３’－位がリン酸基またはチオリン酸基である第二のオリゴヌクレオチドまたはその改変体の複数が、複数のテンプレート核酸をもちいて、環状に連結される、先行する項目のいずれか一項に記載の方法。
(項目４４)
　前記テンプレート核酸が前記第一のオリゴヌクレオチドまたはその改変体の５’末端および前記第二のオリゴヌクレオチドまたはその改変体の３’末端と連結したオリゴヌクレオチドまたはその改変体において、該第一のオリゴヌクレオチドまたはその改変体の５’末端と該第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結される、先行する項目のいずれか一項に記載の方法。
(項目４５)
　先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドまたはその改変体または
先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体
を含む、それぞれ該第二のオリゴヌクレオチドまたはその改変体または該第一のオリゴヌクレオチドまたはその改変体を用いて連結オリゴヌクレオチドまたはその改変体を製造するための組成物。
(項目４６)
　項目１に記載の第一のオリゴヌクレオチドまたはその改変体もしくは
項目１に記載の第二のオリゴヌクレオチドまたはその改変体、またはそれらの組み合わせを含む、テンプレート核酸を用いて連結オリゴヌクレオチドを製造するための組成物。
(項目４７)
　オリゴヌクレオチドまたはその改変体を連結するためのキットであって、
先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドまたはその改変体と、
先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体と、
活性化剤と、
テンプレート核酸と
を含むキット。
(項目４８)
　先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドまたはその改変体および
先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体
を含む、連結オリゴヌクレオチドを製造するための原料組成物。
(項目４９)
　前記第二のオリゴヌクレオチドまたはその改変体を製造するための原料が、２’位にフルオロ基、５’位にアミノ基を有するヌクレオシドを含む、先行する項目のいずれか一項に記載の原料組成物。
(項目５０)
　第一および／または第二のオリゴヌクレオチドまたはその改変体の原料（モノマーまたは置換基導入試薬等）を含む、
先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドもしくはその改変体、および／または
先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドもしくはその改変体を製造するための組成物。
(項目５１)
　前記第一および／または第二のオリゴヌクレオチドまたはその改変体の原料が、アミダイド体のまたはデオキシヌクレオシド、または３’位が水酸基であるまたはデオキシヌクレオシドである、先行する項目のいずれか一項に記載の組成物。
(項目５２)
Ａ）合成を所望する核酸配列の情報を得る工程、
Ｂ）Ａ）の情報に基づき、合成したい配列の一部を複数合成する工程であって、ここで、各配列は、先行する項目のいずれか一項に記載の特徴を有する、工程、ならびに
Ｃ）必要に応じて連結反応に必要な試薬の存在下で、連結条件に配置する工程
を包含する、所望の核酸配列を有するオリゴヌクレオチドまたはその改変体の製造方法。
(項目５３)
Ａ）合成を所望する核酸配列の情報を得る工程、
Ｂ）Ａ）の情報に基づき、合成したい配列の一部を複数合成する工程であって、ここで、各配列は、先行する項目のいずれか一項に記載の特徴を有する、工程、ならびに
Ｃ）必要に応じて連結反応に必要な試薬と、Ｂ）で合成された部分配列を含む核酸とをパッケージとする工程
を包含する、所望のオリゴヌクレオチドまたはその改変体の製造キットの製造方法。
(項目５４)
　以下の式

（式中、Ｙは、水素基またはアルコキシ基であり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ｘは、ヒドロキシ基、またはハロゲンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｎは１以上の整数であり、ｐは１以上の整数であり、Ｒ１は５’－位が水酸基である核酸、もしくは核酸誘導体、またはＲ１は２’－位が水素基またはアルコキシ基であり、５’－位がアミノ基である核酸、もしくは核酸誘導体であり、Ｒ２は３’－位が水酸基、リン酸基もしくはチオリン酸基である核酸、もしくは核酸誘導体、またはＲ２は２’－位がヒドロキシ基、またはハロゲンであり、３’－位がリン酸基もしくはチオリン酸基である核酸、もしくは核酸誘導体であるか、Ｒ１およびＲ２は、一緒になって単結合を形成してもよく、Ｒ１およびＲ２は、異なる化合物のＲ１およびＲ２と結合してもよく、Ｒ３が置換されていてもよいアミノ基である）で示される化合物。
(項目５５)
　前記Ｙは、メトキシ基であり、前記Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、前記Ｘは、フルオロ基であり、Ｒ１およびＲ２は、一緒になって単結合を形成している、先行する項目のいずれか一項に記載の化合物。
(項目５６)
　ｎが５から１０００の整数であり、ｐが５から１０００の整数である、先行する項目のいずれか一項に記載の化合物。
(項目５７)
　前記Ｒ３が、置換されていないアミノ基である、先行する項目のいずれか一項に記載の化合物。
(項目５８)
　前記Ｒ３が、Ｃ１－６アルキル基によって置換されたアミノ基である、先行する項目のいずれか一項に記載の化合物。
(項目５９)
　前記Ｒ３がメチル基またはエチル基によって置換されたアミノ基である、先行する項目のいずれか一項に記載の化合物。
(項目６０)
　前記Ｂが、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、またはウラシルである、先行する項目のいずれか一項に記載の化合物。
(項目６１)
　前記Ｒ１および前記Ｒ２が、一緒になって単結合を形成している、先行する項目のいずれか一項に記載の化合物。
(項目６２)
　先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端と先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体の複数と、前記第一のオリゴヌクレオチドまたはその改変体と、前記第二のオリゴヌクレオチドまたはその改変体とが、連結されている、連結オリゴヌクレオチドまたはその改変体。
(項目６３)
　先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端および先行する項目のいずれか一項に記載のテンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体と連結されている、連結オリゴヌクレオチドまたはその改変体。
(項目６４)
　先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端および先行する項目のいずれか一項に記載のテンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、先行する項目のいずれか一項に記載のオリゴヌクレオチドまたはその改変体と連結されている、連結オリゴヌクレオチドまたはその改変体。
(項目６５)
　先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端と先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体と、前記第一のオリゴヌクレオチドまたはその改変体の３’末端と、第二のオリゴヌクレオチドまたはその改変体の５’末端とが、連結されている、連結オリゴヌクレオチドまたはその改変体。
(項目６６)
　先行する項目のいずれか一項に記載のテンプレート核酸が先行する項目のいずれか一項に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端および先行する項目のいずれか一項に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端と連結したオリゴヌクレオチドまたはその改変体において、該第一のオリゴヌクレオチドまたはその改変体の５’末端と該第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結されている、連結オリゴヌクレオチドまたはその改変体。
(項目６７)
　先行する項目のいずれか一項に記載の化合物を含む、医薬組成物。 Although not intended to be limiting, the present disclosure makes it possible to chemically bind chemically modified RNA molecules with high efficiency, making it possible to create long-chain RNAs with new functions (as a representative diagram, 1). Furthermore, since this reaction is applicable not only to RNA but also to synthetic DNA, it provides an effective technique for producing functional DNA.
The present invention provides, for example, the following items.
(Item 1)
A method for linking a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof, the method comprising:
arranging the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof under conditions in which linkage occurs;
A linkage occurs between the 3' end of the first oligonucleotide or its variant and the 5' end of the second oligonucleotide or its variant, and the terminal monomer portion on the 5' end side of the second oligonucleotide is containing at least one modification;
Method.
(Item 2)
The method according to any one of the preceding items, wherein the 3' end of the first oligonucleotide or variant thereof comprises a phosphate group or a thiophosphate group.
(Item 3)
The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof comprises an optionally substituted amino group.
(Item 4)
The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof comprises an optionally substituted amino group at the 5' position of its terminal monomer moiety.
(Item 5)
A method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof comprises an unsubstituted amino group at the 5' position of its terminal monomer moiety.
(Item 6)
A method according to any of the preceding items, wherein the amino group is substituted by a C 1-6 alkyl group.
(Item 7)
A method according to any one of the preceding items, wherein said amino group is substituted by a methyl group or an ethyl group.
(Item 8)
The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof is substituted with halo or -OH at the 2' position of its terminal monomer moiety.
(Item 9)
The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof is substituted with a halo at the 2' position of its terminal monomer portion.
(Item 10)
The method according to any one of the preceding items, wherein the second oligonucleotide or variant thereof is substituted with a fluoro group at the 2' position of its terminal monomer moiety.
(Item 11)
The method according to any one of the preceding items, wherein the first oligonucleotide or its variant is substituted with a hydrogen group or an optionally substituted alkoxy group at the 2' position of its terminal monomer moiety. .
(Item 12)
The method according to any one of the preceding items, wherein the first oligonucleotide or variant thereof is substituted with an optionally substituted alkoxy at the 2' position of its terminal monomer moiety.
(Item 13)
The method according to any one of the preceding items, wherein the first oligonucleotide or variant thereof is substituted with methoxy at the 2' position of its terminal monomer moiety.
(Item 14)
A method according to any one of the preceding items, wherein the 2' position of the terminal monomer moiety is substituted upwards.
(Item 15)
A method according to any one of the preceding items, wherein the 2' position of the terminal monomer moiety is substituted downward.
(Item 16)
The method according to any one of the preceding items, wherein the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof are linked via a phosphate group or a thiophosphate group. .
(Item 17)
The method of any one of the preceding items, wherein the conditions under which the ligation occurs includes using a template nucleic acid.
(Item 18)
According to any one of the preceding items, the template nucleic acid has a sequence and chain length that are continuous and complementary to the first oligonucleotide or its variant and the second oligonucleotide or its variant. the method of.
(Item 19)
The method according to any one of the preceding items, wherein the template nucleic acid has a chain length of 10 residues or more.
(Item 20)
The method according to any one of the preceding items, wherein the template nucleic acid has a chain length of 20 to 40 residues.
(Item 21)
The template nucleic acid is not linked to the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof, or either one of the oligonucleotides or a variant thereof, or both. A method according to any one of the preceding items, wherein the method is linked to an oligonucleotide or a variant thereof.
(Item 22)
The sequence of the template nucleic acid includes a sequence complementary to the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof, or mismatched base pairs within a range that does not prevent the formation of double strands. , the method described in any one of the preceding items.
(Item 23)
A method according to any of the preceding items, wherein the conditions under which the ligation occurs includes the use of an activator of the oligonucleotide ligation reaction.
(Item 24)
The first oligonucleotide or its variant has the following formula (I)

(In the formula, R - is S - or O - , Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine) , 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-amino purine, or 2,6-diaminopurine, A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, n is an integer greater than or equal to 1, and R 1 is a hydroxyl group at the 5'-position. a certain nucleic acid or a nucleic acid derivative)
The method according to any one of the preceding items, wherein the compound is a compound of the formula:
(Item 25)
A method according to any one of the preceding items, wherein said Y is a methoxy group.
(Item 26)
A method according to any one of the preceding items, wherein n is an integer from 5 to 1000.
(Item 27)
The second oligonucleotide or its variant has the following formula (II)

(wherein, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, p is an integer greater than or equal to 1, and R 2 is a nucleic acid in which the 3′-position is a hydroxyl group, a phosphate group, or a thiophosphoric acid group, or a nucleic acid derivative)
The method according to any one of the preceding items, wherein the compound is a compound of the formula:
(Item 28)
A method according to any one of the preceding items, wherein said X is a fluoro group.
(Item 29)
A method according to any one of the preceding items, wherein p is an integer from 5 to 1000.
(Item 30)
A method according to any one of the preceding items, wherein the spacer is an optionally substituted C 1-10 alkylene or PEG.
(Item 31)
A method according to any one of the preceding items, wherein the spacer is a C3 alkylene.
(Item 32)
A method according to any one of the preceding items, wherein the activating agent is a carbodiimide condensing agent, a phosphonium condensing agent, a uronium condensing agent or a triazine condensing agent.
(Item 33)
According to any one of the preceding items, the carbodiimide condensing agent is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC), diisopropylcarbodiimide (DIPCI) or dicyclohexylcarbodiimide (DCC). the method of.
(Item 34)
The phosphonium condensing agent is bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), 1H-benzotriazol-1-yloxytris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyBOP), 6-trifluoro Methyl-1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyFOP), 1H-benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) or (7-aza Benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP).
(Item 35)
The uronium-based condensing agent is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazole-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroboric acid salt (TSTU) or {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate (COMU). The method described in paragraph 1.
(Item 36)
A method according to any one of the preceding items, wherein the triazine-based condensing agent is DMT-MM.
(Item 37)
Any one of the preceding items, wherein the activator is 1-fluoro-2,4-dinitrobenzene (DNFB) or 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC). The method described in section.
(Item 38)
The activator may be 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt) or N- A method according to any one of the preceding items, used in combination with hydroxysuccinimide (HOSu).
(Item 39)
a plurality of oligonucleotides or variants thereof, in which the 5' end of the first oligonucleotide or variant thereof and the 3' end of the second oligonucleotide or variant thereof, and the first oligonucleotide or variant thereof; The method according to any one of the preceding items, wherein the variant and the second oligonucleotide or variant thereof are linked at two or more locations simultaneously or stepwise.
(Item 40)
Any of the preceding items, wherein the oligonucleotide or variant thereof to which the 5' end of the first oligonucleotide or variant thereof and the template nucleic acid are linked is linked to the second oligonucleotide or variant thereof. The method described in paragraph 1.
(Item 41)
Any of the preceding items, wherein the oligonucleotide or variant thereof to which the 3' end of the second oligonucleotide or variant thereof and the template nucleic acid are linked is linked to the first oligonucleotide or variant thereof. The method described in paragraph 1.
(Item 42)
An oligonucleotide or a variant thereof in which the 5' end of the first oligonucleotide or a variant thereof is linked to the 3' end of a second oligonucleotide or a variant thereof, and the first oligonucleotide or a variant thereof. and the 5' end of the second oligonucleotide or variant thereof are linked.
(Item 43)
Any one of the preceding items, wherein a plurality of second oligonucleotides or variants thereof in which the 3'-position of R2 is a phosphate group or a thiophosphate group are linked in a circular manner using a plurality of template nucleic acids. The method described in section.
(Item 44)
In the oligonucleotide or a variant thereof, in which the template nucleic acid is linked to the 5' end of the first oligonucleotide or the variant thereof and the 3' end of the second oligonucleotide or the variant thereof, the first oligonucleotide or the variant thereof, and the 3' end of the second oligonucleotide or variant thereof are linked.
(Item 45)
a first oligonucleotide according to any one of the preceding items or a variant thereof or a second oligonucleotide according to any one of the preceding items or a variant thereof, respectively; A composition for producing a linked oligonucleotide or a variant thereof using a nucleotide or a variant thereof, or the first oligonucleotide or a variant thereof.
(Item 46)
For producing a linked oligonucleotide using a template nucleic acid comprising the first oligonucleotide according to item 1 or a variant thereof, the second oligonucleotide according to item 1 or a variant thereof, or a combination thereof. Composition.
(Item 47)
A kit for linking oligonucleotides or variants thereof, comprising:
a first oligonucleotide according to any one of the preceding items or a variant thereof;
a second oligonucleotide according to any one of the preceding items or a variant thereof;
an activator;
A kit comprising a template nucleic acid.
(Item 48)
Producing a linked oligonucleotide comprising a first oligonucleotide according to any one of the preceding items or a variant thereof and a second oligonucleotide according to any one of the preceding items or a variant thereof Raw material composition for.
(Item 49)
The raw material composition according to any one of the preceding items, wherein the raw material for producing the second oligonucleotide or a variant thereof contains a nucleoside having a fluoro group at the 2' position and an amino group at the 5' position. thing.
(Item 50)
Containing raw materials (monomers or substituent introduction reagents, etc.) for the first and/or second oligonucleotides or variants thereof;
A composition for producing a first oligonucleotide according to any one of the preceding items or a variant thereof and/or a second oligonucleotide according to any one of the preceding items or a variant thereof. thing.
(Item 51)
According to any one of the preceding items, the raw material for the first and/or second oligonucleotide or a variant thereof is an amidide or a deoxynucleoside, or a deoxynucleoside in which the 3' position is a hydroxyl group. Composition of.
(Item 52)
A) obtaining information on the nucleic acid sequence desired to be synthesized;
B) A step of synthesizing a plurality of parts of the sequence to be synthesized based on the information in A), where each sequence has the characteristics described in any one of the preceding items, and C ) A method for producing an oligonucleotide having a desired nucleic acid sequence or a variant thereof, which comprises the step of placing the oligonucleotide in ligation conditions, optionally in the presence of reagents necessary for the ligation reaction.
(Item 53)
A) obtaining information on the nucleic acid sequence desired to be synthesized;
B) A step of synthesizing a plurality of parts of the sequence to be synthesized based on the information in A), where each sequence has the characteristics described in any one of the preceding items, and C ) A method for producing a kit for producing a desired oligonucleotide or a variant thereof, which includes the step of packaging, if necessary, a reagent necessary for the ligation reaction and a nucleic acid containing the partial sequence synthesized in B).
(Item 54)
The following formula

(In the formula, Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine , 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine. , X is a hydroxy group or a halogen, A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, n is an integer of 1 or more, p is an integer of 1 or more, R 1 is a nucleic acid or a nucleic acid derivative in which the 5'-position is a hydroxyl group, or R 1 is a nucleic acid or a nucleic acid derivative in which the 2'-position is a hydrogen group or an alkoxy group and the 5'-position is an amino group. , R 2 is a nucleic acid or a nucleic acid derivative whose 3'-position is a hydroxyl group, phosphate group, or thiophosphoric acid group, or R 2 is a hydroxyl group or a halogen at the 2'-position and a phosphoric acid group at the 3'-position. R 1 and R 2 may be taken together to form a single bond, and R 1 and R 2 are R 1 and R 2 of different compounds. 2 and R 3 is an optionally substituted amino group.
(Item 55)
Said Y is a methoxy group, and said B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine. , pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine, and the X is , a fluoro group, and R 1 and R 2 are taken together to form a single bond.
(Item 56)
A compound according to any one of the preceding items, wherein n is an integer from 5 to 1000 and p is an integer from 5 to 1000.
(Item 57)
A compound according to any one of the preceding items, wherein R 3 is an unsubstituted amino group.
(Item 58)
A compound according to any one of the preceding items, wherein R 3 is an amino group substituted by a C 1-6 alkyl group.
(Item 59)
A compound according to any one of the preceding items, wherein R 3 is an amino group substituted by a methyl group or an ethyl group.
(Item 60)
A compound according to any one of the preceding items, wherein B is each independently adenine, cytosine, methylcytosine, guanine, thymine, or uracil.
(Item 61)
A compound according to any one of the preceding items, wherein said R 1 and said R 2 are taken together to form a single bond.
(Item 62)
The 5' end of the first oligonucleotide or variant thereof according to any one of the preceding items and the 3' end of the second oligonucleotide or variant thereof according to any one of the preceding items. A linked oligonucleotide or a variant thereof, in which a plurality of linked oligonucleotides or variants thereof, the first oligonucleotide or a variant thereof, and the second oligonucleotide or a variant thereof are linked. .
(Item 63)
The 5' end of the first oligonucleotide or variant thereof according to any one of the preceding items and the oligonucleotide or variant thereof linked to the template nucleic acid according to any one of the preceding items, A linked oligonucleotide or a variant thereof, which is linked to a second oligonucleotide or a variant thereof according to any one of the items.
(Item 64)
The 3' end of the second oligonucleotide or variant thereof according to any one of the preceding items and the oligonucleotide or variant thereof linked to the template nucleic acid according to any one of the preceding items, A linked oligonucleotide or a variant thereof, which is linked to the oligonucleotide or variant thereof according to any one of the items.
(Item 65)
The 5' end of the first oligonucleotide or variant thereof according to any one of the preceding items and the 3' end of the second oligonucleotide or variant thereof according to any one of the preceding items. A linked oligonucleotide or a variant thereof, the 3' end of the first oligonucleotide or variant thereof, and the 5' end of the second oligonucleotide or variant thereof are linked. Nucleotides or variants thereof.
(Item 66)
the 5' end of the first oligonucleotide or variant thereof according to any one of the preceding items, which is preceded by the template nucleic acid according to any one of the preceding items; In an oligonucleotide or a variant thereof linked to the 3' end of a second oligonucleotide or variant thereof, the 5' end of the first oligonucleotide or variant thereof and the 5' end of the second oligonucleotide or variant thereof. A linked oligonucleotide or a variant thereof, which is linked to the 3' end.
(Item 67)
A pharmaceutical composition comprising a compound according to any one of the preceding items.
　本開示において、上記１または複数の特徴は、明示された組み合わせに加え、さらに組み合わせて提供されうることが意図される。本開示のなおさらなる実施形態および利点は、必要に応じて以下の詳細な説明を読んで理解すれば、当業者に認識される。 In the present disclosure, it is intended that the one or more features described above may be provided in further combinations in addition to the specified combinations. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, as appropriate.
　長鎖のオリゴヌクレオチドを合成することができるオリゴヌクレオチドの化学的連結反応を提供する。本連結反応を用いることにより、これまでの化学的連結反応よりも収率が向上することができる。 Provides a chemical ligation reaction for oligonucleotides that can synthesize long-chain oligonucleotides. By using this ligation reaction, the yield can be improved compared to conventional chemical ligation reactions.
図１は、オリゴヌクレオチド連結反応の概略図を示す。５’ｏｌｉｇｏの３’末端のＭと３’ｏｌｉｇｏの５’末端のＱを結合させ、２本のｏｌｉｇｏ鎖を連結する。ＯｌｉｇｏはＲＮＡ、ＤＮＡまたはその混合物、いずれでも良い。Ｍは３’－リン酸から誘導される基（Ｒ＝Ｓ，Ｏ）を有し、Ｑは５’－アミノ基を有する。FIG. 1 shows a schematic diagram of the oligonucleotide ligation reaction. The M at the 3' end of the 5' oligo and the Q at the 5' end of the 3' oligo are linked to link the two oligo chains. Oligo may be RNA, DNA, or a mixture thereof. M has a group derived from 3'-phosphoric acid (R=S,O) and Q has a 5'-amino group. 図２は、オリゴヌクレオチド連結反応の反応スキームを示す。５’ｏｌｉｇｏとして、Ｍのヌクレオチド残基中の２’位としてＹ＝メトキシ基、水素、フルオロ基のｏｌｉｇｏを、３’ｏｌｉｇｏとして、Ｑのヌクレオチド残基中の２’位としてＸ＝水酸基、フルオロ基のオリゴヌクレオチド鎖を用いる。FIG. 2 shows a reaction scheme of oligonucleotide ligation reaction. As a 5'oligo, an oligo with Y = methoxy group, hydrogen, or fluoro group as the 2' position in the nucleotide residue of M, and as a 3' oligo, an oligo with X = hydroxyl group, fluoro group as the 2' position in the nucleotide residue of Q. A base oligonucleotide chain is used. 図３は、オリゴヌクレオチド連結反応に用いたオリゴヌクレオチドまたは核酸の配列を示す。FIG. 3 shows the sequences of oligonucleotides or nucleic acids used in oligonucleotide ligation reactions. 図４は、Qに2’F-5’-NH2-ウリジンを導入するための試薬（5’-アミノ-2’-フルオロ-2’,5’-ジデオキシウリジン　アミダイト試薬）の合成スキームを示す。Figure 4 shows a synthesis scheme of a reagent (5'-amino-2'-fluoro-2',5'-dideoxyuridine amidite reagent) for introducing 2'F-5'-NH 2 -uridine into Q. . 図５は、Qに2’OH-5’-NH2-ウリジンを導入するための試薬（5’-アミノ-5’-デオキシウリジン　アミダイト試薬）の構造を示す。FIG. 5 shows the structure of a reagent (5'-amino-5'-deoxyuridine amidite reagent) for introducing 2'OH-5'-NH 2 -uridine into Q. 図６は、2FpsとDNFBとの反応のHPLCによる分析を示す。Figure 6 shows HPLC analysis of the reaction between 2Fps and DNFB. 図７は、ＤＮＦＢ修飾した５’－ｏｌｉｇｏと３’－ｏｌｉｇｏの反応のＰＡＧＥによる分析を示す。（Ａ）ＲＮＡをテンプレートとしてＤＮＦＢ修飾した５’－ｏｌｉｇｏと３’－ｏｌｉｇｏを反応し、１０，３０，６０，１２０分後のサンプルを分析（Ｂ）２Ｍｅｐｓ－ＤＮＦＢと２ＯＨ５Ｎおよび２Ｆ５Ｎの反応率の経時変化（Ｃ）２Ｆｐｓ－ＤＮＦＢと２ＯＨ５Ｎおよび２Ｆ５Ｎの反応率の経時変化を示す。FIG. 7 shows PAGE analysis of the reaction between DNFB-modified 5'-oligo and 3'-oligo. (A) DNFB-modified 5'-oligo and 3'-oligo were reacted using RNA as a template, and samples were analyzed after 10, 30, 60, and 120 minutes. (B) Reaction rate of 2Meps-DNFB, 2OH5N, and 2F5N Change over time (C) Shows the change over time in the reaction rate of 2Fps-DNFB, 2OH5N, and 2F5N. 図８は、ＤＮＦＢ修飾した５’－ｏｌｉｇｏと３’－ｏｌｉｇｏの１２０ｍｉｎの反応における反応率の比較を示す。FIG. 8 shows a comparison of reaction rates in a 120 min reaction between DNFB-modified 5'-oligo and 3'-oligo. 図９は、５’－ｏｌｉｇｏと３’－ｏｌｉｇｏのＤＮＦＢ存在下、６時間反応のＰＡＧＥによる分析を示す。（Ａ）ＲＮＡテンプレートを利用した反応の２Ｍｅｐｓと２Ｆｐｓの比較、（Ｂ）ＲＮＡおよびＤＮＡテンプレートを利用した反応の２ＯＨ５Ｎと２Ｆ５Ｎの比較を示す。FIG. 9 shows PAGE analysis of a 6-hour reaction of 5'-oligo and 3'-oligo in the presence of DNFB. (A) Comparison of 2Meps and 2Fps in reactions using RNA templates, and (B) Comparison of 2OH5N and 2F5N in reactions using RNA and DNA templates. 図１０は、５’－ｏｌｉｇｏと３’－ｏｌｉｇｏのＥＤＣ存在下での反応を示す。（Ａ）５’ｏｌｉｇｏと３’ｏｌｉｇｏの６０ｍｉｎの反応における反応率の比較、（Ｂ）２ＯＨ５Ｎと２Ｍｅｐｏの連結体のＨＰＬＣによる分析、（Ｃ）２Ｆ５Ｎと２Ｍｅｐｏの連結体のＨＰＬＣによる分析を示す。FIG. 10 shows the reaction of 5'-oligo and 3'-oligo in the presence of EDC. (A) Comparison of reaction rates in 60 min reaction of 5'oligo and 3'oligo, (B) HPLC analysis of 2OH5N and 2Mepo conjugate, (C) HPLC analysis of 2F5N and 2Mepo conjugate. 図１１は、テンプレートのＤＮＡを用いずに、お互いの鎖がテンプレートになるように作製する連結反応の模式図を示す。FIG. 11 shows a schematic diagram of a ligation reaction in which each strand serves as a template without using template DNA. 図１２は、Ｑに２’－Ｆ－５’－ＮＨ２　シチジンを導入するための試薬（５’－アミノ－２’－フルオロ－２’，５’－ジデオキシウリジン　アミダイト試薬、化合物５）の合成スキームを示す。Figure 12 shows the synthesis of a reagent (5'-amino-2'-fluoro - 2',5'-dideoxyuridine amidite reagent, compound 5) for introducing 2'-F-5'-NH2cytidine into Q. Show the scheme. 図１３は、Ｑに２’－ＯＨ－５’－ＮＨ２　シチジンを導入するための試薬（５’－アミノ－５’－デオキシシチジン　アミダイト試薬、化合物１１）の構造を示す。Figure 13 shows the structure of a reagent for introducing 2'-OH-5'-NH 2 cytidine into Q (5'-amino-5'-deoxycytidine amidite reagent, compound 11). 図１４は、５’－ｏｌｉｇｏと３’－ｏｌｉｇｏのＤＮＦＢ存在下、６時間反応のＰＡＧＥによる分析を示す。ＲＮＡおよびＤＮＡテンプレートを利用した反応の２ＯＨ５Ｎ＿Ｃと２Ｆ５Ｎ＿Ｃの比較を示す。FIG. 14 shows PAGE analysis of a 6-hour reaction of 5'-oligo and 3'-oligo in the presence of DNFB. A comparison of 2OH5N_C and 2F5N_C reactions using RNA and DNA templates is shown. 図１５は、５’－ｏｌｉｇｏと３’－ｏｌｉｇｏのＥＤＣ存在下での３０ｍｉｎの反応における反応率の比較を示す。FIG. 15 shows a comparison of the reaction rates of 5'-oligo and 3'-oligo in a 30 min reaction in the presence of EDC.
　以下に、本開示をさらに詳細に説明する。
　本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形の冠詞（例えば、英語の場合は「ａ」、「ａｎ」、「ｔｈｅ」等）は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用されるすべての専門用語および科学技術用語は、本開示の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書（定義を含めて）が優先する。 The present disclosure will be described in further detail below.
Throughout this specification, references to the singular should be understood to include the plural unless specifically stated otherwise. Accordingly, singular articles (e.g., "a,""an,""the," etc. in English) should be understood to include the plural concept, unless specifically stated otherwise. Further, it should be understood that the terms used herein have the meanings commonly used in the art unless otherwise specified. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will control.
　（定義）
　本明細書において「基」とは、別途指定されない限り、一価基を意味する。一価基でない例としては、アルキレン基（２価）等が挙げられる。また、下記の置換基等の説明において、「基」なる用語を省略する場合もある。 (definition)
As used herein, "group" means a monovalent group unless otherwise specified. Examples of non-monovalent groups include alkylene groups (divalent) and the like. Furthermore, in the following description of substituents, etc., the term "group" may be omitted in some cases.
　本明細書において、「必要に応じて置換された」もしくは「置換されている」で定義される場合における置換基の数は、特に限定がない場合、置換可能であれば特に制限はなく、１または複数である。また、特に指示した場合を除き、各々の置換基の説明はその置換基が他の置換基の一部分または置換基である場合にも該当する。 In the present specification, the number of substituents when defined as "optionally substituted" or "substituted" is not particularly limited as long as it is substitutable, unless there is a particular limitation; or more than one. Further, unless otherwise specified, the description of each substituent also applies when the substituent is a part of or a substituent of another substituent.
　本明細書において「上向きで置換」とは、結合される環に対して上方向に結合が出るように置換されていることをいう。核酸（ヌクレオシド）の場合、糖部のフラノース環（５員環）を平面とした時に、核酸塩基と同じ側に置換されていることをいう。 As used herein, "substituted upward" refers to substitution such that the bond is directed upward with respect to the ring to which it is bonded. In the case of nucleic acids (nucleosides), when the furanose ring (5-membered ring) of the sugar moiety is viewed as a plane, it is substituted on the same side as the nucleobase.
　本明細書において「下向きで置換」とは、結合される環に対して下方向に結合が出るように置換されていることをいう。核酸（ヌクレオシド）の場合、糖部のフラノース環（５員環）を平面とした時に、核酸塩基と逆側に置換されていることをいう。 As used herein, "substituted downward" refers to substitution such that the bond is directed downward with respect to the ring to which it is bonded. In the case of nucleic acids (nucleosides), when the furanose ring (5-membered ring) of the sugar moiety is viewed as a plane, it is substituted on the opposite side to the nucleobase.
　本明細書において「必要に応じて置換された」もしくは「置換されている」で修飾されている基において、該基のいずれの部分が置換されていてもよい。例えば、「必要に応じて置換されたアリールアルキル」および「置換されているアリールアルキル」は、アリール部分が置換されていても、アルキル部分が置換されていてもよく、アリール部分およびアルキル部分の両方が置換されていてもよい。 In the group modified with "optionally substituted" or "substituted" in this specification, any part of the group may be substituted. For example, "optionally substituted arylalkyl" and "substituted arylalkyl" may be substituted at the aryl moiety or substituted at the alkyl moiety, and include both the aryl moiety and the alkyl moiety. may be replaced.
　本明細書において「C1-6」とは、炭素原子数が１～６であることを意味する。他の数字の場合も同様であり、例えば、「C1-4」とは炭素原子数が１～４であること、「C1-3」とは炭素原子数が１～３であることを意味する。本明細書において炭素数の限定がある記載はあくまで好ましい数値範囲であり、本開示は規定される炭素数以外の炭素数の置換基を有する基も本開示の範囲内であることが意図される。 As used herein, "C 1-6 " means having 1 to 6 carbon atoms. The same applies to other numbers; for example, "C 1-4 " means that the number of carbon atoms is 1 to 4, and "C 1-3 " means that the number of carbon atoms is 1 to 3. means. In this specification, descriptions in which the number of carbon atoms is limited are only preferred numerical ranges, and the present disclosure is intended to include groups having substituents with a number of carbon atoms other than the specified number of carbon atoms within the scope of the present disclosure. .
　本明細書において「ハロゲン原子」は、ハロゲン族に属する原子であって、フッ素原子、塩素原子、臭素原子またはヨウ素原子等を意味する。好ましくはフッ素原子、又は塩素原子である。さらに好ましくは、フッ素原子である。「ハロゲン原子」を「ハロゲン」、「ハロ」と称する場合もある。 As used herein, the term "halogen atom" refers to an atom belonging to the halogen group, such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. Preferably it is a fluorine atom or a chlorine atom. More preferably, it is a fluorine atom. A "halogen atom" is sometimes referred to as "halogen" or "halo."
　本明細書において「水酸基」は、－ＯＨの一価基である。この基は、「ヒドロキシ基」、「ヒドロキシ」と呼ばれる場合もある。 In this specification, the "hydroxyl group" is a monovalent group of -OH. This group is sometimes referred to as a "hydroxy group" or "hydroxy."
　本明細書において「アミノ」は－ＮＨ２基を指す。 As used herein, "amino" refers to the group -NH2 .
　本明細書において「リン酸基」は

を指す。リン酸基の酸素原子は硫黄原子と置き換えられてもよく、酸素原子が硫黄原子と置き換えられたリン酸基を、本明細書において「チオリン酸基」という。置換される酸素原子は１～４個であり得るが、好ましくは1個である。 In this specification, "phosphate group" is

refers to The oxygen atom of the phosphoric acid group may be replaced with a sulfur atom, and a phosphoric acid group in which the oxygen atom is replaced with a sulfur atom is referred to herein as a "thiophosphoric acid group." The number of oxygen atoms to be substituted may be 1 to 4, but preferably 1.
　本明細書において「アルキル」は、直鎖または分枝鎖状の飽和脂肪族炭化水素基を意味する。「Ｃ１－１２アルキル」は、炭素原子数１～１２のアルキル基であり、例としては、Ｃ１－６アルキル、ヘプチル、イソヘプチル、オクチル、イソオクチル、ノニル、イソノニル、デシル、イソデシル、ウンデシル、イソウンデシル、ドデシル、イソドデシル等が挙げられるが、これらに限定されない。「Ｃ１－６アルキル」は、炭素原子数１～６のアルキル基であり、好ましい例としては、「Ｃ１－４アルキル」が挙げられ、より好ましくは「Ｃ１－３アルキル」であり、さらに好ましくは「Ｃ１－２アルキル」である。「Ｃ１－４アルキル」の具体例としては、メチル、エチル、プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｔｅｒｔ－ブチル、ｓｅｃ－ブチル等が挙げられる。「Ｃ１－６アルキル」の具体例としては、Ｃ１－４アルキル、ｎ－ペンチル、イソペンチル、ネオペンチル、ｔｅｒｔ－ペンチル、１，２－ジメチルプロピル、ｎ－ヘキシル等が挙げられるが、これらに限定されない。 As used herein, "alkyl" refers to a straight or branched saturated aliphatic hydrocarbon group. “C 1-12 alkyl” is an alkyl group having 1 to 12 carbon atoms, and examples thereof include C 1-6 alkyl, heptyl, isoheptyl, octyl, isooctyl, nonyl, isononyl, decyl, isodecyl, undecyl, isoundecyl. , dodecyl, isododecyl and the like, but are not limited to these. "C 1-6 alkyl" is an alkyl group having 1 to 6 carbon atoms, and preferred examples include "C 1-4 alkyl", more preferably "C 1-3 alkyl", More preferred is "C 1-2 alkyl". Specific examples of "C 1-4 alkyl" include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, and the like. Specific examples of "C 1-6 alkyl" include, but are not limited to, C 1-4 alkyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, 1,2-dimethylpropyl, n-hexyl, etc. Not done.
　本明細書において「アルコキシ」は、－Ｏ－アルキルの一価基である。アルコキシの好ましい例としては、Ｃ１－６アルコキシ（即ち、Ｃ１－６アルキル－Ｏ－）、Ｃ１－４アルコキシ（即ち、Ｃ１－４アルキル－Ｏ－）等が挙げられる。Ｃ１－４アルコキシの具体例としては、メトキシ（ＣＨ３Ｏ－）、エトキシ（ＣＨ３ＣＨ２Ｏ－）、ｎ－プロポキシ（ＣＨ３（ＣＨ２）２Ｏ－）、イソプロポキシ（（ＣＨ３）２ＣＨＯ－）、ｎ－ブトキシ（ＣＨ３（ＣＨ２）３Ｏ－）、イソブトキシ（（ＣＨ３）２ＣＨＣＨ２Ｏ－）、ｔｅｒｔ－ブトキシ（（ＣＨ３）３ＣＯ－）、ｓｅｃ－ブトキシ（ＣＨ３ＣＨ２ＣＨ（ＣＨ３）Ｏ－）等が挙げられる。Ｃ１－６アルコキシの具体例としては、Ｃ１－４アルコキシ、ｎ－ペンチルオキシ（ＣＨ３（ＣＨ２）４Ｏ－）、イソペンチルオキシ（（ＣＨ３）２ＣＨＣＨ２ＣＨ２Ｏ－）、ネオペンチルオキシ（（ＣＨ３）３ＣＣＨ２Ｏ－）、ｔｅｒｔ－ペンチルオキシ（ＣＨ３ＣＨ２Ｃ（ＣＨ３）２Ｏ－）、１，２－ジメチルプロポキシ（ＣＨ３ＣＨ（ＣＨ３）ＣＨ（ＣＨ３）Ｏ－）等が挙げられるが、これらに限定されない。 As used herein, "alkoxy" is a monovalent group of -O-alkyl. Preferred examples of alkoxy include C 1-6 alkoxy (ie, C 1-6 alkyl-O-), C 1-4 alkoxy (ie, C 1-4 alkyl-O-), and the like. Specific examples of C 1-4 alkoxy include methoxy (CH 3 O-), ethoxy (CH 3 CH 2 O-), n-propoxy (CH 3 (CH 2 ) 2 O-), isopropoxy ((CH 3 ) 2 CHO-), n-butoxy (CH 3 (CH 2 ) 3 O-), isobutoxy ((CH 3 ) 2 CHCH 2 O-), tert-butoxy ((CH 3 ) 3 CO-), sec-butoxy (CH 3 CH 2 CH(CH 3 )O-) and the like. Specific examples of C 1-6 alkoxy include C 1-4 alkoxy, n-pentyloxy (CH 3 (CH 2 ) 4 O-), isopentyloxy ((CH 3 ) 2 CHCH 2 CH 2 O-), Neopentyloxy ((CH 3 ) 3 CCH 2 O-), tert-pentyloxy (CH 3 CH 2 C(CH 3 ) 2 O-), 1,2-dimethylpropoxy (CH 3 CH(CH 3 )CH( Examples include, but are not limited to, CH 3 )O-) and the like.
　本明細書において「ヌクレオチド」および「ヌクレオチド分子」とは、糖部分がリン酸エステルになっているヌクレオシドをいう。本明細書において「ヌクレオシド」とは、塩基と糖とがＮ－グリコシド結合をした化合物をいう。糖部分がＤ－リボースのものをリボヌクレオチドといい、ＲＮＡの加水分解によって得られる。糖部分がＤ－２’－デオキシリボースのものをデオキシリボヌクレオチドといい、ＤＮＡの酵素分解によって得られる。本明細書において、ヌクレオチドは、天然のものでも非天然のものでもよく、人工的に合成された物でもよい。「ヌクレオチド」は改変されてもよく、改変は、塩基、糖部分およびリン酸基部分のいずれかあるいはその組み合わせにおいて施され得、好ましくはヌクレオチドの糖部分、リン酸基部分が修飾され得る、および／またはヌクレオチドの３’位がリン酸基で修飾され得る。ヌクレオチドの改変体において、ヌクレオチドの３’位がリン酸基で修飾され、リン酸基の少なくとも一つの酸素原子が硫黄原子に置換され得る。ヌクレオチドの改変体の代表例は式（１）に示される化合物のモノマー部分であり得るがこれに限定されない。 As used herein, "nucleotide" and "nucleotide molecule" refer to a nucleoside in which the sugar moiety is a phosphate ester. As used herein, "nucleoside" refers to a compound in which a base and a sugar form an N-glycosidic bond. Ribonucleotides whose sugar moiety is D-ribose are obtained by hydrolysis of RNA. Those whose sugar moiety is D-2'-deoxyribose are called deoxyribonucleotides, which are obtained by enzymatic degradation of DNA. In this specification, nucleotides may be natural or non-natural, or may be artificially synthesized. A "nucleotide" may be modified, and the modification may be made in any one or a combination of the base, sugar moiety, and phosphate moiety, preferably the sugar moiety, the phosphate moiety of the nucleotide, and /or the 3' position of the nucleotide may be modified with a phosphate group. In nucleotide variants, the 3' position of the nucleotide may be modified with a phosphate group, and at least one oxygen atom of the phosphate group may be replaced with a sulfur atom. A representative example of a nucleotide variant may be, but is not limited to, the monomer portion of the compound represented by formula (1).
　本明細書において「オリゴヌクレオチド」および「ポリヌクレオチド」は、交換可能に使用され、ヌクレオチドのポリマーまたはオリゴマーを指す。「オリゴヌクレオチド」を「oligo」、「核酸」または「核酸鎖」と称する場合もある。「オリゴヌクレオチド」は改変されてもよく、改変は、塩基、糖部分およびリン酸基部分のいずれかあるいはその組み合わせにおいて施され得、好ましくはオリゴヌクレオチドの糖部分、リン酸基部分が修飾され得る、および／またはオリゴヌクレオチドの３’末端の３’位がリン酸基で修飾され得る。オリゴヌクレオチドの改変体において、オリゴヌクレオチドの３’末端の３’位がリン酸基で修飾され、リン酸基の少なくとも一つの酸素原子が硫黄原子に置換され得る。本明細書において「オリゴヌクレオチドの改変体」および「ポリヌクレオチドの改変体」は、交換可能に使用され、ヌクレオチドおよび／またはヌクレオチドの改変体のポリマーまたはオリゴマーを指す。オリゴヌクレオチドの改変体の代表例は式（１）に示される化合物であるがこれに限定されない。本明細書の文脈において、当該ポリマーまたはオリゴマーにおいて、１つでもヌクレオチドの改変体を含む場合オリゴヌクレオチドの改変体またはポリヌクレオチドの改変体という。本明細書においてオリゴヌクレオチドおよびオリゴヌクレオチドの改変体は、２以上のヌクレオチドまたはその改変体が重合している限りその長さに限定はなく、その他の文脈で制限があると解されない限り、限定されない。なお、この数は、本明細書においては塩基の数として計数されることもある。 "Oligonucleotide" and "polynucleotide" are used interchangeably herein and refer to a polymer or oligomer of nucleotides. An "oligonucleotide" may also be referred to as "oligo," "nucleic acid," or "nucleic acid strand." The "oligonucleotide" may be modified, and the modification may be made in any one or a combination of the base, sugar moiety, and phosphate group, and preferably the sugar moiety and the phosphate group of the oligonucleotide may be modified. , and/or the 3' position of the 3' end of the oligonucleotide may be modified with a phosphate group. In a variant of the oligonucleotide, the 3' position of the 3' end of the oligonucleotide may be modified with a phosphate group, and at least one oxygen atom of the phosphate group may be replaced with a sulfur atom. As used herein, "oligonucleotide variant" and "polynucleotide variant" are used interchangeably and refer to a polymer or oligomer of nucleotides and/or nucleotide variants. A typical example of a modified oligonucleotide is a compound represented by formula (1), but is not limited thereto. In the context of this specification, if the polymer or oligomer contains at least one nucleotide variant, it is referred to as an oligonucleotide variant or a polynucleotide variant. As used herein, oligonucleotides and oligonucleotide variants are not limited in length as long as two or more nucleotides or variants thereof are polymerized, and unless there is a limitation in other contexts. . Note that this number may be counted as the number of bases in this specification.
　本明細書において、「デオキシリボ核酸（ＤＮＡ）」は少なくとも１つのデオキシリボヌクレオチドモノマー部分を含む分子を意味する。 As used herein, "deoxyribonucleic acid (DNA)" refers to a molecule containing at least one deoxyribonucleotide monomer moiety.
　本明細書において、「リボ核酸（ＲＮＡ）」は少なくとも１つのリボヌクレオチドモノマー部分を含む分子を意味する。「リボヌクレオチド」とは、β－Ｄ－リボ－フラノース部分の２’位においてヒドロキシル基を有するヌクレオチドを意味する。 As used herein, "ribonucleic acid (RNA)" refers to a molecule that includes at least one ribonucleotide monomer moiety. "Ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribo-furanose moiety.
　本明細書において、核酸の文脈において使用される「改変」（「修飾」ともいい英語ではいずでもｍｏｄｉｆｉｃａｔｉｏｎ／ｍｏｄｉｆｙ、ｓｕｂｓｔｉｔｕｔｉｏｎ、ｓｕｂｓｔｉｔｕｔｅ、ｄｉｓｐｌａｃｅｍｅｎｔ、ｄｉｓｐｌａｃｅ、ｒｅｐｌａｃｅｍｅｎｔ、ｒｅｐｌａｃｅという。）とは、核酸の構成単位またはその末端の一部または全部が他の原子団と置換されること、または官能基が付加されている状態を指す。そのような「改変」が含まれるものを（ＤＮＡ、ＲＮＡ、核酸またはオリゴヌクレオチドなどの）「改変体」という。オリゴヌクレオチドの改変体としては、メチル化等のアルキル化が可能なものはすべて列挙することができ、本明細書において開示される任意の改変（種々の置換基による改変）を含むものが挙げられ、このほか、当該分野では例えば、ＢＮＡ（Ｂｒｉｄｇｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ）、ＬＮＡ（Ｌｏｃｋｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ）等を挙げることができ、このような人工核酸などの改変体についてはWengel　et　al.,Eur.J.Org.Chem.2297-2321(2005)を参照されたい。この文献は、本明細書において参考として援用される。 As used herein, "modification" (also referred to as "modification" in English) used in the context of nucleic acids refers to the structure of nucleic acids. Refers to a state in which a part or all of the unit or its terminal is substituted with another atomic group, or a functional group is added. A substance (DNA, RNA, nucleic acid, oligonucleotide, etc.) containing such a "modification" is referred to as a "variant". Modified oligonucleotides include all oligonucleotides that can be alkylated such as methylation, and include any modifications disclosed herein (modifications with various substituents). , In addition, in this field, for example, BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), etc. can be mentioned, and modifications of such artificial nucleic acids are described in Wengel et al., Eur.J.Org. Please refer to .Chem.2297-2321(2005). This document is incorporated herein by reference.
　本明細書において「核酸塩基」または「塩基」とは、核酸を構成する塩基成分をいい、たとえば、これに限定されるわけではないがアデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）、チミン（Ｔ）、ウラシル（Ｕ）、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、２，６－ジアミノプリン等が挙げられる。 As used herein, the term "nucleobase" or "base" refers to a base component constituting a nucleic acid, including, but not limited to, adenine (A), guanine (G), cytosine (C), Thymine (T), uracil (U), hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, Examples include 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, and 2,6-diaminopurine.
　本明細書において「末端モノマー部分」とは、オリゴヌクレオチドの５’末端または３’末端にある部分（代表的には、ヌクレオシド部分）をいう。末端モノマー部分は、改変されていてもよく、例えば、２’位がフルオロ基で置換されていてもよい。 As used herein, the term "terminal monomer portion" refers to a portion (typically a nucleoside portion) at the 5' or 3' end of an oligonucleotide. The terminal monomer moiety may be modified, for example, the 2' position may be substituted with a fluoro group.
　本明細書において「テンプレート核酸」とは、オリゴヌクレオチドまたはその改変体と相補的な配列を有する核酸をいう。 As used herein, the term "template nucleic acid" refers to a nucleic acid having a sequence complementary to an oligonucleotide or a variant thereof.
本明細書において「オリゴヌクレオチドまたはその改変体の３’末端」とは、オリゴヌクレオチドまたはその改変体の３’側に結合している末端モノマー部分をいう。 As used herein, the term "3' end of an oligonucleotide or a variant thereof" refers to the terminal monomer portion bound to the 3' side of the oligonucleotide or a variant thereof.
　本明細書において「オリゴヌクレオチドまたはその改変体の５’末端」とは、オリゴヌクレオチドまたはその改変体の５’側に結合している末端モノマー部分をいう。 As used herein, "the 5' end of an oligonucleotide or a variant thereof" refers to the terminal monomer portion bonded to the 5' side of the oligonucleotide or a variant thereof.
　本明細書において「連結」とは、任意の化学的結合やリンカーなどを介して結びついていることを意味し、該化学的結合としては、これに限定するものではないが、例えば共有結合、イオン結合、水素結合などを含む。好ましくは本開示の連結は、共有結合である。 In this specification, "linking" means being connected via any chemical bond or linker, etc., and the chemical bonds include, but are not limited to, covalent bonds, ionic bonds, etc. Including bonds, hydrogen bonds, etc. Preferably the linkage of the present disclosure is a covalent bond.
　本明細書において「連結が生じる条件」とはオリゴヌクレオチドまたはその改変体を連結することができる条件をいう。本開示において「連結が生じる条件」はリガーゼなどの核酸連結活性を有する酵素を使用しない条件が想定される。したがって、連結が生じる条件は、温度、ｐＨ、溶媒、塩濃度（バッファー、塩化ナトリウム等）などが挙げられ適宜当業者によって調整され得る。 As used herein, "conditions under which ligation occurs" refers to conditions under which oligonucleotides or variants thereof can be ligated. In the present disclosure, "conditions under which ligation occurs" are assumed to be conditions in which an enzyme having nucleic acid ligation activity such as ligase is not used. Therefore, conditions for ligation include temperature, pH, solvent, salt concentration (buffer, sodium chloride, etc.), and can be appropriately adjusted by those skilled in the art.
　本明細書において「連続して相補的な配列および鎖長」とは、第一のオリゴヌクレオチドまたはその改変体の３’末端と第二のオリゴヌクレオチドまたはその改変体の５’末端とが、連結が生じた際できるオリゴヌクレオチドまたはその改変体の第一のオリゴヌクレオチドまたはその改変体部分の一部から連結部分を含んで第二のオリゴヌクレオチドまたはその改変体部分の一部の配列と相補的な配列およびオリゴヌクレオチドまたはその改変体部分の一部から連結部分を含んで第二のオリゴヌクレオチドまたはその改変体部分の一部の配列の配列を含めるような相補的な配列を形成できる鎖長をいう。鎖長は、第一のオリゴヌクレオチドおよび第二のオリゴヌクレオチドと連続して二本鎖を形成できる範囲であれば、長くても短くてよく。好ましくは１０残基以上であり、より好ましくは２０～４０残基である。 As used herein, "continuously complementary sequence and chain length" means that the 3' end of the first oligonucleotide or its variant and the 5' end of the second oligonucleotide or its variant are linked. is generated when an oligonucleotide or its variant is complementary to the sequence of a portion of a second oligonucleotide or its variant portion, including a linking portion, from a portion of the first oligonucleotide or its variant portion. Refers to the chain length that can form a complementary sequence from a sequence and a part of an oligonucleotide or its variant part, including a linking part, to the sequence of a part of a second oligonucleotide or its variant part. . The chain length may be long or short as long as it can continuously form a double strand with the first oligonucleotide and the second oligonucleotide. It is preferably 10 or more residues, more preferably 20 to 40 residues.
　本明細書において「二本鎖の形成を妨げない範囲」とは、連結反応を行う条件（反応の温度等）においてオリゴヌクレオチドまたはその改変体とテンプレート核酸が二本鎖を形成することができる範囲をいう。 As used herein, "the range that does not prevent the formation of double strands" refers to the range in which the oligonucleotide or its variant and the template nucleic acid can form a double strand under the conditions in which the ligation reaction is carried out (reaction temperature, etc.). means.
　本明細書において「二本鎖の形成を妨げない範囲のミスマッチ塩基対」とは、相補的な配列でないミスマッチ塩基対が、オリゴヌクレオチドまたはその改変体とテンプレート核酸が二本鎖を形成することができる範囲にあるミスマッチ塩基対をいう。 As used herein, "mismatched base pairs within a range that does not prevent the formation of double strands" refers to mismatched base pairs that are not complementary sequences that do not allow the oligonucleotide or its variant and template nucleic acid to form a double strand. Refers to mismatched base pairs within the possible range.
　本明細書において「活性化剤」とは、化合物を活性化して、反応を活性化し、反応速度および反応収率を向上すことができるものをいい、特に3’末端のリン酸から誘導される基を活性化して、オリゴヌクレオチドまたはその改変体のモノマー部分との間でホスホロアミデート結合を形成させることができるものをいう。例えば、カルボジイミド系縮合剤、ホスホニウム系縮合剤、ウロニウム系縮合剤またはトリアジン系縮合剤が挙げられる。 As used herein, the term "activator" refers to an agent capable of activating a compound, activating the reaction, and improving the reaction rate and reaction yield. A group that can be activated to form a phosphoramidate bond with a monomer portion of an oligonucleotide or a variant thereof. Examples include carbodiimide condensing agents, phosphonium condensing agents, uronium condensing agents, and triazine condensing agents.
　本明細書において「オリゴヌクレオチド連結反応の活性化剤」とは、オリゴヌクレオチドまたはその改変体を連結する際に用いることができる活性剤をいう。例えば、カルボジイミド系縮合剤、ホスホニウム系縮合剤、ウロニウム系縮合剤またはトリアジン系縮合剤が挙げられる。 As used herein, the term "activator for oligonucleotide ligation reaction" refers to an activator that can be used when ligating oligonucleotides or variants thereof. Examples include carbodiimide condensing agents, phosphonium condensing agents, uronium condensing agents, and triazine condensing agents.
　本明細書において「核酸誘導体」とは、天然に存在する核酸とは異なるがもとの核酸と同様の機能を有するものをいう。核酸の塩基、糖、リン酸ジエステル部に化学修飾を加えたものも含む。例えば、ホスホロチオエート核酸、ＬＮＡ修飾核酸、２’－ＯＭｅ修飾核酸、２’－Ｆ修飾核酸、２’－ＭＯＥ修飾核酸、ＰＭＯ核酸、ＰＮＡ核酸等が挙げられる。 As used herein, the term "nucleic acid derivative" refers to a derivative that is different from a naturally occurring nucleic acid but has a similar function to the original nucleic acid. It also includes chemically modified nucleic acids in the base, sugar, and phosphodiester moieties. Examples include phosphorothioate nucleic acids, LNA-modified nucleic acids, 2'-OMe-modified nucleic acids, 2'-F-modified nucleic acids, 2'-MOE-modified nucleic acids, PMO nucleic acids, PNA nucleic acids, and the like.
　本明細書において「カルボジイミド系縮合剤」とは分子内にカルボジイミドを含む縮合剤をいう。例えば、１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ（水溶性カルボジイミド（water　soluble　carbodiimide(ＷＳＣ、ＷＳＣＤ）とも呼ばれる））、ジイソプロピルカルボジイミド（ＤＩＰＣＩ）またはジシクロヘキシルカルボジイミド（ＤＣＣ）等が挙げられる。 As used herein, the term "carbodiimide condensing agent" refers to a condensing agent containing carbodiimide in the molecule. For example, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC (also called water soluble carbodiimide (WSC, WSCD)), diisopropylcarbodiimide (DIPCI), dicyclohexylcarbodiimide (DCC), etc. can be mentioned.
　本明細書において「ホスホニウム系縮合剤」とは分子内にホスホニウムを含む縮合剤をいう。例えば、ブロモトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＢｒｏＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ピロリジン－１－イル）ホスホニウム・ヘキサフルオロホスファート（ＰｙＢＯＰ）、６－トリフルオロメチル－１Ｈ－ベンゾトリアゾール－１－イルオキシトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＦＯＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ジメチルアミノ）　ホスホニウムヘキサフルオロホスファート（ＢＯＰ）または（７－アザベンゾトリアゾール－１－イルオキシ）トリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＡＯＰ）等が挙げられる。 As used herein, the term "phosphonium-based condensing agent" refers to a condensing agent containing phosphonium in the molecule. For example, bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), 1H-benzotriazol-1-yloxytris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyBOP), 6-trifluoromethyl-1H- Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyFOP), 1H-benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) or (7-azabenzotriazole-1 -yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP) and the like.
　本明細書において「ウロニウム系縮合剤」とは分子内にウロニウムを含む縮合剤をいう。例えば、Ｏ－（７－アザベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（ＨＡＴＵ）、Ｏ－（ベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（ＨＢＴＵ）、Ｏ－（Ｎ－スクシンイミジル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウム　テトラフルオロほう酸塩（ＴＳＴＵ）または｛｛［（１－シアノ－２－エトキシ－２－オキソエチリデン）アミノ］オキシ｝－４－モルホリノメチレン｝ジメチルアンモニウム　ヘキサフルオロホスファート（ＣＯＭＵ）等が挙げられる。 As used herein, the term "uronium-based condensing agent" refers to a condensing agent containing uronium in the molecule. For example, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N , N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU) or {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate (COMU).
　本明細書において「トリアジン系縮合剤」とは分子内にトリアジンを含む縮合剤をいう。例えば、ＤＭＴ－ＭＭ等が挙げられる。 As used herein, the term "triazine-based condensing agent" refers to a condensing agent containing triazine in the molecule. For example, DMT-MM and the like can be mentioned.
　本明細書において「アミダイド体のヌクレオシドまたはデオキシヌクレオシド」とはヌクレオシドまたはデオキシヌクレオシドの３’位がリン酸エステルの構造を有する化合物をいう。 As used herein, the term "amidide nucleoside or deoxynucleoside" refers to a compound in which the 3' position of a nucleoside or deoxynucleoside has a phosphate ester structure.
　本明細書において「キット」とは、通常２つ以上の区画に分けて、提供されるべき部分が提供されるユニットをいう。安定性等のため、混合されて提供されるべきでなく、使用直前に混合して使用することが好ましいような組成物の提供を目的とするときに、このキットの形態は好ましい。 As used herein, the term "kit" refers to a unit in which parts to be provided are usually divided into two or more compartments. This kit format is preferable when the purpose is to provide a composition that should not be provided mixed for reasons of stability etc., but is preferably mixed immediately before use.
　本明細書において「原料」とは、オリゴヌクレオチドまたはその改変体を連結させる際に用いる物質をいう。例えば、モノマーまたは置換基導入試薬等が挙げられ、好ましくはアミダイド体のまたはデオキシヌクレオシド、または３’位が水酸基であるまたはデオキシヌクレオシドである。 As used herein, "raw material" refers to a substance used when linking oligonucleotides or variants thereof. Examples include monomers or substituent-introducing reagents, and preferred are amidite or deoxynucleosides, or deoxynucleosides in which the 3' position is a hydroxyl group.
　本明細書において用いられる場合、「疾患」（ｄｉｓｅａｓｅ）、「障害」（ｄｉｓｏｒｄｅｒ）および「状態」もしくは「症状」（いずれもｃｏｎｄｉｔｉｏｎ）は交換可能に用いられる。 As used herein, "disease", "disorder", and "state" or "symptom" (all conditions) are used interchangeably.
　　（好ましい実施形態）
　以下に本開示の好ましい実施形態を説明する。以下に提供される実施形態は、本開示のよりよい理解のために提供されるものであり、本開示の範囲は以下の記載に限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参酌して、本開示の範囲内で適宜改変を行うことができることは明らかである。また、本開示の以下の実施形態は単独でも使用されあるいはそれらを組み合わせて使用することができることが理解される。
　（オリゴヌクレオチドまたはその改変体の連結） (Preferred embodiment)
Preferred embodiments of the present disclosure will be described below. It is understood that the embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can take the description in this specification into account and make appropriate modifications within the scope of the present disclosure. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.
(Linkage of oligonucleotide or variant thereof)
　１つの局面において、本開示は、第一のオリゴヌクレオチドまたはその改変体と第二のオリゴヌクレオチドまたはその改変体とを連結させる方法であって、第一のオリゴヌクレオチドまたはその改変体と第二のオリゴヌクレオチドまたはその改変体とを、連結が生じる条件下に配置する工程を含み、第一のオリゴヌクレオチドまたはその改変体の３’末端と、第二のオリゴヌクレオチドまたはその改変体の５’末端において連結が生じ、第二のオリゴヌクレオチドまたはその改変体の５’末端側の末端モノマー部分が少なくとも一つの改変を含む方法を提供する。 In one aspect, the present disclosure provides a method for linking a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof, the method comprising: linking a first oligonucleotide or a variant thereof to a second oligonucleotide or a variant thereof; the step of placing the oligonucleotide or a variant thereof under conditions in which ligation occurs, at the 3' end of the first oligonucleotide or variant thereof and the 5' end of the second oligonucleotide or variant thereof. A method is provided in which ligation occurs and the terminal monomer portion at the 5' end of the second oligonucleotide or variant thereof comprises at least one modification.
　本開示の技術に従って、第一のオリゴヌクレオチドまたはその改変体と、第二のオリゴヌクレオチドまたはその改変体とを連結させる技術を用いることで、オリゴヌクレオチドまたはその改変体間の反応性の向上、高い反応収率を達成することができる。また、本開示の技術を用いることによって、新たな機能を有した長鎖ＲＮＡの作製が可能になり、ＲＮＡのみならず合成ＤＮＡに対しても適用可能なことから、機能性ＤＮＡの作製も行うことができる。 According to the technology of the present disclosure, by using the technique of linking the first oligonucleotide or its variant and the second oligonucleotide or its variant, the reactivity between the oligonucleotides or their variants can be improved and reaction yields can be achieved. Furthermore, by using the technology of the present disclosure, it is possible to produce long-chain RNA with new functions, and since it is applicable not only to RNA but also to synthetic DNA, functional DNA will also be produced. be able to.
　一実施形態において、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体は、置換されていてもよいアミノ基を含み、好ましくはその末端モノマー部分の５’位に置換されていてもよいアミノ基を含む。理論に束縛されることを望まないが、アミノ基または置換アミノ基を有するオリゴヌクレオチドまたはその改変体を用いることで、オリゴヌクレオチドまたはその改変体間の反応性の向上、高い反応収率を達成することができる。 In one embodiment, the second oligonucleotide or variant thereof used in the present disclosure comprises an optionally substituted amino group, preferably an optionally substituted amino group at the 5' position of its terminal monomer moiety. Contains groups. Without wishing to be bound by theory, it is possible to improve the reactivity between oligonucleotides or their variants and achieve high reaction yields by using oligonucleotides or variants thereof having amino groups or substituted amino groups. be able to.
　一実施形態において、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体に含まれるアミノ基は、置換されていないアミノ基である。 In one embodiment, the amino group included in the second oligonucleotide or variant thereof used in the present disclosure is an unsubstituted amino group.
　一実施形態において、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体に含まれるアミノ基は、Ｃ１－６アルキル基によって置換されており、例示的には、本開示において用いられるアミノ基は、メチル基またはエチル基によって置換されている。 In one embodiment, the amino group included in the second oligonucleotide or variant thereof used in the present disclosure is substituted with a C 1-6 alkyl group, illustratively, the amino group used in the present disclosure is substituted with a methyl or ethyl group.
　一実施形態において、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がハロまたは－ＯＨで置換されていて、好ましくは、その末端残基の２’位がハロで置換されている。より好ましくは、その末端モノマー部分の２’位がフルオロ基で置換されている。理論に束縛されることを望まないが、末端モノマー部分の２’位が置換されることで、オリゴヌクレオチドまたはその改変体間の反応性の向上、高い反応収率を達成することができる。 In one embodiment, the second oligonucleotide or variant thereof used in the present disclosure is substituted with halo or -OH at the 2' position of its terminal monomer moiety, preferably with a halo or -OH at the 2' position of its terminal monomer moiety; The position is replaced by halo. More preferably, the 2' position of the terminal monomer moiety is substituted with a fluoro group. Although not wishing to be bound by theory, by substituting the 2'-position of the terminal monomer moiety, it is possible to improve the reactivity between oligonucleotides or their variants and achieve a high reaction yield.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位が水素基または置換されていてもよいアルコキシ基で置換されていて、好ましくは、その末端モノマー部分の２’位が置換されていてもよいアルコキシで置換されている。より好ましくは、その末端モノマー部分の２’位がメトキシで置換されている。理論に束縛されることを望まないが、末端モノマー部分の２’位が置換されることで、オリゴヌクレオチドまたはその改変体間の反応性の向上、高い反応収率を達成することができる。 In one embodiment, the first oligonucleotide used in the present disclosure or a variant thereof is substituted with a hydrogen group or an optionally substituted alkoxy group at the 2' position of the terminal monomer moiety, and preferably, The 2' position of the terminal monomer moiety is substituted with optionally substituted alkoxy. More preferably, the 2' position of the terminal monomer moiety is substituted with methoxy. Although not wishing to be bound by theory, by substituting the 2'-position of the terminal monomer moiety, it is possible to improve the reactivity between oligonucleotides or their variants and achieve a high reaction yield.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体の末端モノマー部分の２’位は上向きまたは下向きで置換されている。好ましくは第一のオリゴヌクレオチドまたはその改変体の末端モノマー部分の２’位は下向きで置換されている。 In one embodiment, the 2' position of the terminal monomer portion of the first oligonucleotide used in this disclosure or a variant thereof is substituted upward or downward. Preferably, the 2' position of the terminal monomer portion of the first oligonucleotide or variant thereof is substituted downward.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体の少なくとも１つにおけるリン酸基は、Ｓ－またはＯ－を含む。 In one embodiment, the phosphate group in at least one of the first oligonucleotides or variants thereof used in this disclosure comprises S - or O - .
　一実施形態において、本開示において用いられる前記第一のオリゴヌクレオチドまたはその改変体の３’末端は、リン酸基またはチオリン酸基を含む。 In one embodiment, the 3' end of the first oligonucleotide or variant thereof used in the present disclosure includes a phosphate group or a thiophosphate group.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体と前記第二のオリゴヌクレオチドまたはその改変体が、リン酸基またはチオリン酸基を介して連結している。 In one embodiment, the first oligonucleotide or its variant used in the present disclosure and the second oligonucleotide or its variant are linked via a phosphate group or a thiophosphate group.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体と前記第二のオリゴヌクレオチドまたはその改変体の複数が連結される。複数の本開示のオリゴヌクレオチドまたはその改変体を用いることで、オリゴヌクレオチドまたはその改変体の鎖長が数百あるいは1000以上のオリゴヌクレオチドまたはその改変体を連結することができる。 In one embodiment, the first oligonucleotide or a variant thereof used in the present disclosure and a plurality of the second oligonucleotides or variants thereof are linked. By using a plurality of oligonucleotides or variants thereof of the present disclosure, oligonucleotides or variants thereof having chain lengths of several hundred or 1000 or more can be linked.
　一実施形態において、本開示において用いられる連結が生じる条件は、テンプレート核酸を用いることを包含する。好ましくはテンプレート核酸は、第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体と連続して相補的な配列および鎖長を有する。ここで第一のオリゴヌクレオチドまたはその改変体が５’から３’末端に向かってＧＣＵＧＡＡＧＧＧＣの塩基配列を有し、第二のオリゴヌクレオチドまたはその改変体が５’から３’末端に向かってＵＵＵＵＧＡＡＣＵＣＵＧＣの塩基配列を有する場合、テンプレート核酸の連続して相補的な配列は５’から３’末端に向かってＵＣＡＡＡＡＧＣＣＣＵＵとなる。 In one embodiment, the conditions for ligation used in this disclosure include using a template nucleic acid. Preferably, the template nucleic acid has a sequence and length that is continuous and complementary to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof. Here, the first oligonucleotide or its variant has a base sequence of GCUGAAGGGGC from 5' to 3' end, and the second oligonucleotide or its variant has a base sequence of UUUUGAACUCUGC from 5' to 3' end. In the case where the template nucleic acid has a continuous complementary sequence from the 5' to the 3' end, the sequence is UCAAAAGCCCUU.
　一実施形態において、本開示において用いられるテンプレート核酸は、第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体とは連結していないか、または、いずれか一方のオリゴヌクレオチドまたはその改変体、もしくは両方のオリゴヌクレオチドまたはその改変体と連結している。好ましくは、本開示において用いられるテンプレート核酸は、第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体とは連結していない。 In one embodiment, the template nucleic acid used in this disclosure is not linked to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof, or to either one of the oligonucleotides or or a variant thereof, or both oligonucleotides or a variant thereof. Preferably, the template nucleic acid used in this disclosure is not linked to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof.
　一実施形態において、本開示において用いられるテンプレート核酸の配列は、第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体と相補的な配列、あるいは二本鎖の形成を妨げない範囲のミスマッチ塩基対を含む。 In one embodiment, the sequence of the template nucleic acid used in this disclosure is a sequence that is complementary to the first oligonucleotide or variant thereof and the second oligonucleotide or variant thereof, or does not prevent duplex formation. Contains a range of mismatched base pairs.
　一実施形態において、本開示において用いられる連結が生じる条件は、提供されるオリゴヌクレオチドまたはその改変体が、テンプレート核酸などと共存したときに連結が進む条件であれば、どのような条件であってもよく、他の薬剤の存在、温度、ｐＨ、溶媒、塩濃度（バッファー、塩化ナトリウム等）などを適宜当業者は決定することができる。好ましくは、オリゴヌクレオチドまたはその改変体連結反応の活性化剤が用いられる。 In one embodiment, the conditions for ligation used in the present disclosure are any conditions as long as the ligation proceeds when the provided oligonucleotide or its variant coexists with a template nucleic acid, etc. Those skilled in the art can appropriately determine the presence of other drugs, temperature, pH, solvent, salt concentration (buffer, sodium chloride, etc.), etc. Preferably, an activator for the ligation reaction of oligonucleotides or variants thereof is used.
　一実施形態において、本開示において用いられる活性化剤は、カルボジイミド系縮合剤、ホスホニウム系縮合剤、ウロニウム系縮合剤またはトリアジン系縮合剤であり、好ましくは、カルボジイミド系縮合剤としては１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）、ジイソプロピルカルボジイミド（ＤＩＰＣＩ）を挙げることができ、ホスホニウム系縮合剤としては、ブロモトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＢｒｏＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ピロリジン－１－イル）ホスホニウム・ヘキサフルオロホスファート（ＰｙＢＯＰ）、６－トリフルオロメチル－１Ｈ－ベンゾトリアゾール－１－イルオキシトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＦＯＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ジメチルアミノ）　ホスホニウムヘキサフルオロホスファート（ＢＯＰ）または（７－アザベンゾトリアゾール－１－イルオキシ）トリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＡＯＰ）を挙げることができる。また、ウロニウム系縮合剤としてはＯ－（７－アザベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（ＨＡＴＵ）、Ｏ－（ベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－　テトラメチルウロニウムヘキサフルオロホスファート（ＨＢＴＵ）、Ｏ－（Ｎ－スクシンイミジル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウム　テトラフルオロほう酸塩（ＴＳＴＵ）または｛｛［（１－シアノ－２－エトキシ－２－オキソエチリデン）アミノ］　オキシ｝－４－モルホリノメチレン｝ジメチルアンモニウム　ヘキサフルオロホスファート（ＣＯＭＵ）を挙げることができ、また、トリアジン系縮合剤としてはＤＭＴ－ＭＭを挙げることができるが、これらに限定されない。 In one embodiment, the activating agent used in the present disclosure is a carbodiimide-based condensing agent, a phosphonium-based condensing agent, a uronium-based condensing agent, or a triazine-based condensing agent, and preferably the carbodiimide-based condensing agent is 1-[3 -(dimethylamino)propyl]-3-ethylcarbodiimide (EDC), diisopropylcarbodiimide (DIPCI), and examples of the phosphonium condensing agent include bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), 1H-benzo Triazol-1-yloxytris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyBOP), 6-trifluoromethyl-1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyFOP) , 1H-benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP). can. In addition, examples of uronium-based condensing agents include O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetra Mention may be made of fluoroborate (TSTU) or {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate (COMU), and Examples of the triazine condensing agent include, but are not limited to, DMT-MM.
　一実施形態において、本開示において用いられる活性化剤は、１－フルオロ－２，４－ジニトロベンゼン（ＤＮＦＢ）、または１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）である。水中での連結反応に用いることができる、水中で使用可能な活性化剤が好ましい。 In one embodiment, the activating agent used in the present disclosure is 1-fluoro-2,4-dinitrobenzene (DNFB), or 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC). be. Preferred are activators that can be used in water and can be used in ligation reactions in water.
　一実施形態において、本開示において用いられる活性化剤は、１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）と１－ヒドロキシ－７－アザベンゾトリアゾール（ＨＯＡｔ）、１－ヒドロキシベンゾトリアゾール（ＨＯＢｔ）またはＮ－ヒドロキシコハク酸イミド（ＨＯＳｕ）とを組み合わせて使用される。 In one embodiment, the activators used in the present disclosure include 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt), 1- It is used in combination with hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (HOSu).
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体は、下記式（Ｉ）

（式中、Ｒ－はＳ－またはＯ－であり、Ｙは水素基またはアルコキシ基であり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｎは１以上の整数であり、Ｒ１は５’－位が水酸基である核酸、または核酸誘導体である）で示される化合物である。理論に束縛されることを望まないが、このような特定の構造を用いることで、新たな機能を有した長鎖ＲＮＡの作製が可能になり、ＲＮＡのみならず合成ＤＮＡに対しても適用可能なことから、機能性ＤＮＡの作製することができる。理論に束縛されることを望まないが、末端モノマー部分の２’位が置換されることで、オリゴヌクレオチドまたはその改変体間の反応性の向上、高い反応収率を達成することができる。 In one embodiment, the first oligonucleotide used in the present disclosure or a variant thereof has the following formula (I):

(In the formula, R - is S - or O - , Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine) , 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-amino purine, or 2,6-diaminopurine, A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, n is an integer greater than or equal to 1, and R 1 is a hydroxyl group at the 5'-position. A compound represented by a certain nucleic acid or a nucleic acid derivative). Without wishing to be bound by theory, the use of such a specific structure makes it possible to create long RNAs with new functions, and can be applied not only to RNA but also to synthetic DNA. Therefore, functional DNA can be produced. Although not wishing to be bound by theory, by substituting the 2'-position of the terminal monomer moiety, it is possible to improve the reactivity between oligonucleotides or their variants and achieve a high reaction yield.
　一実施形態において、第一のオリゴヌクレオチドまたはその改変体におけるＹはメトキシ基である。 In one embodiment, Y in the first oligonucleotide or a variant thereof is a methoxy group.
　一実施形態において、ｎは５から１０００の整数である。好ましくはｎは５から９００の整数、ｎは５から８００の整数、ｎは５から７００の整数、ｎは５から６００の整数、ｎは５から５００の整数、ｎは５から４００の整数、ｎは５から３００の整数、ｎは５から２００の整数、ｎは５から１００の整数であり、より好ましくは５から２００の整数である。特により好ましくは１０から１００の整数である。 In one embodiment, n is an integer from 5 to 1000. Preferably n is an integer from 5 to 900, n is an integer from 5 to 800, n is an integer from 5 to 700, n is an integer from 5 to 600, n is an integer from 5 to 500, n is an integer from 5 to 400, n is an integer from 5 to 300, n is an integer from 5 to 200, n is an integer from 5 to 100, and more preferably an integer from 5 to 200. Particularly more preferred is an integer from 10 to 100.
　一実施形態において、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体が、下記式（ＩＩ）

（式中、Ｘは、ヒドロキシ基またはハロゲンであり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｐは１以上の整数であり、Ｒ２は３’－位が水酸基またはリン酸基またはチオリン酸基である核酸、または核酸誘導体である）
で示される化合物である。 In one embodiment, the second oligonucleotide used in the present disclosure or a variant thereof has the following formula (II):

(wherein, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, p is an integer greater than or equal to 1, and R2 is a nucleic acid whose 3'-position is a hydroxyl group, a phosphate group, or a thiophosphate group, or is a nucleic acid derivative)
This is a compound represented by
　一実施形態において、第二のオリゴヌクレオチドまたはその改変体におけるＸはフルオロ基である。理論に束縛されることを望まないが、Ｘとしてフルオロ基を採用することにより、オリゴヌクレオチドまたはその改変体間の反応性の向上、高い反応収率の改善がみられ得る。 In one embodiment, X in the second oligonucleotide or variant thereof is a fluoro group. Although not wishing to be bound by theory, by employing a fluoro group as X, it is possible to see an improvement in the reactivity between oligonucleotides or their variants and a high reaction yield.
　一実施形態において、ｐは５から１０００の整数である。好ましくはｐは５から９００の整数、ｐは５から８００の整数、ｐは５から７００の整数、ｐは５から６００の整数、ｐは５から５００の整数、ｐは５から４００の整数、ｐは５から３００の整数、ｐは５から２００の整数、ｐは５から１００の整数であり、より好ましくは１０から１００の整数である。 In one embodiment, p is an integer from 5 to 1000. Preferably p is an integer from 5 to 900, p is an integer from 5 to 800, p is an integer from 5 to 700, p is an integer from 5 to 600, p is an integer from 5 to 500, p is an integer from 5 to 400, p is an integer from 5 to 300, p is an integer from 5 to 200, p is an integer from 5 to 100, and more preferably an integer from 10 to 100.
　一実施形態において、本開示において用いられるスペーサーは、C１-ｋアルキレンまたはＰＥＧであり、好ましく置換されていてもよいＣ１－１０アルキレンである。いくつかの実施形態においてスペーサーは置換されていてもよいＣ１－１０アルキレンである。いくつかの実施形態においてスペーサーは－（ＣＨ２）ｇ－であり、ｇは１～１０の整数である。いくつかの実施形態においてｇは１である。いくつかの実施形態においてｇは２である。いくつかの実施形態においてｇは３である。いくつかの実施形態においてｇは４である。いくつかの実施形態においてｇは５である。いくつかの実施形態においてｇは６である。いくつかの実施形態においてｇは７である。いくつかの実施形態においてｇは８である。いくつかの実施形態においてｇは９である。いくつかの実施形態においてｇは１０である。好ましくは、ｇは３であり、二本鎖形成における構造および不安定化への影響が最小になる。 In one embodiment, the spacer used in this disclosure is C 1-k alkylene or PEG, preferably optionally substituted C 1-10 alkylene. In some embodiments, the spacer is an optionally substituted C 1-10 alkylene. In some embodiments, the spacer is -(CH 2 ) g -, where g is an integer from 1 to 10. In some embodiments g is 1. In some embodiments g is 2. In some embodiments g is 3. In some embodiments g is 4. In some embodiments g is 5. In some embodiments g is 6. In some embodiments g is 7. In some embodiments g is 8. In some embodiments g is 9. In some embodiments g is 10. Preferably, g is 3 to minimize structural and destabilizing effects on duplex formation.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体の５’末端と本開示において用いられる第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体の複数と、前記第一のオリゴヌクレオチドまたはその改変体と、前記第二のオリゴヌクレオチドまたはその改変体とは、２か所以上同時にまたは段階的に連結される。この場合、同時とは一度の反応条件において連結させることをいい、段階的とは同時ではなく、順次なされることをいう。３つ以上の分子が利用される場合は、各々同時であっても段階的であってもよく、それらの組み合わせであってもよい。 In one embodiment, an oligonucleotide or an oligonucleotide thereof in which the 5' end of the first oligonucleotide used in the present disclosure or a variant thereof and the 3' end of the second oligonucleotide used in the present disclosure or a variant thereof are linked. The plurality of variants, the first oligonucleotide or its variant, and the second oligonucleotide or its variant are linked at two or more locations simultaneously or in stages. In this case, "simultaneously" refers to linking under one reaction condition, and "stepwise" refers to linking not simultaneously but sequentially. When three or more molecules are used, they may be used simultaneously, in stages, or in a combination thereof.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体の５’末端および本開示において用いられるテンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体と連結される。テンプレート核酸と第一のオリゴヌクレオチドまたはその改変体が連結していることにより、別途テンプレート核酸を加えることなく連結反応を行うことができる。 In one embodiment, the oligonucleotide or variant thereof to which the 5' end of the first oligonucleotide used in the present disclosure or a variant thereof and the template nucleic acid used in the present disclosure are linked is the oligonucleotide or variant thereof used in the present disclosure. linked to an oligonucleotide or a variant thereof. By linking the template nucleic acid and the first oligonucleotide or its variant, the linking reaction can be performed without adding a separate template nucleic acid.
　一実施形態において、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体の３’末端および本開示において用いられるテンプレート核酸が連結したオリゴヌクレオチドまたはその改変体分子が、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体と連結される。テンプレート核酸と第二のオリゴヌクレオチドまたはその改変体が連結していることにより、別途テンプレート核酸を加えることなく連結反応を行うことができる。 In one embodiment, the oligonucleotide or variant thereof used in the present disclosure is linked to the 3' end of the second oligonucleotide or variant thereof used in the present disclosure and the template nucleic acid used in the present disclosure. oligonucleotide or a variant thereof. By linking the template nucleic acid and the second oligonucleotide or a variant thereof, the linking reaction can be performed without adding a separate template nucleic acid.
　一実施形態において、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体の５’末端と第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体と、本開示において用いられる第一のオリゴヌクレオチドまたはその改変体の３’末端と、本開示において用いられる第二のオリゴヌクレオチドまたはその改変体の５’末端とが、連結される。第二のオリゴヌクレオチドまたはその改変体とテンプレート核酸と第二のオリゴヌクレオチドまたはその改変体が連結していることにより、環状オリゴヌクレオチドまたはその改変体を合成することができる。 In one embodiment, an oligonucleotide or a variant thereof in which the 5' end of the first oligonucleotide or variant thereof used in the present disclosure is linked to the 3' end of the second oligonucleotide or variant thereof; The 3' end of the first oligonucleotide or variant thereof used in the disclosure and the 5' end of the second oligonucleotide or variant thereof used in the present disclosure are linked. By linking the second oligonucleotide or its variant, the template nucleic acid, and the second oligonucleotide or its variant, a cyclic oligonucleotide or its variant can be synthesized.
　一実施形態において、Ｒ２の３’－位がリン酸基またはチオリン酸基である第二のオリゴヌクレオチドまたはその改変体の複数が、複数のテンプレート核酸をもちいて、環状に連結される。この結果、得られるオリゴヌクレオチドまたはその改変体は環状となり、環状オリゴヌクレオチドまたはその改変体を生成することができる。 In one embodiment, a plurality of second oligonucleotides or variants thereof in which the 3'-position of R 2 is a phosphate group or a thiophosphate group are linked in a circle using a plurality of template nucleic acids. As a result, the obtained oligonucleotide or its variant becomes circular, and a cyclic oligonucleotide or its variant can be produced.
　一実施形態において、本開示において用いられるテンプレート核酸が前記第一のオリゴヌクレオチドまたはその改変体の５’末端および本開示において用いられる第二のオリゴヌクレオチドまたはその改変体の３’末端と連結したオリゴヌクレオチドまたはその改変体において、第一のオリゴヌクレオチドまたはその改変体の５’末端と第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結される。 In one embodiment, the template nucleic acid used in the present disclosure is an oligonucleotide linked to the 5' end of the first oligonucleotide or variant thereof and the 3' end of the second oligonucleotide or variant thereof used in the present disclosure. In the nucleotide or its variant, the 5' end of the first oligonucleotide or its variant and the 3' end of the second oligonucleotide or its variant are linked.
　１つの局面において、本開示は、テンプレート核酸の存在下、３’末端にリン酸から誘導される基を有する第一のオリゴヌクレオチドまたはその改変体と、５’末端に５’－アミノ－５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体とを、３’末端のリン酸から誘導される基を活性化して、５’－アミノ－５’－デオキシヌクレオチドモノマー部分との間でホスホロアミデート結合を形成させることで２つのオリゴヌクレオチド断片を連結させるオリゴヌクレオチドの連結反応において、２’－位にフルオロ基を導入した５’－アミノ－２’－フルオロ－２’，５’－ジデオキシヌクレオチドモノマー部分を用いて行うオリゴヌクレオチドの連結反応を提供する。 In one aspect, the present disclosure provides a first oligonucleotide or a variant thereof having a group derived from phosphate at the 3' end and a 5'-amino-5' at the 5' end in the presence of a template nucleic acid. - a second oligonucleotide having a deoxynucleotide monomer moiety or a variant thereof, by activating a group derived from phosphate at the 3' end, and a 5'-amino-5'-deoxynucleotide monomer moiety; In an oligonucleotide ligation reaction in which two oligonucleotide fragments are linked by forming a phosphoroamidate bond, 5'-amino-2'-fluoro-2',5, which has a fluoro group introduced at the 2'-position, Oligonucleotide ligation reactions performed using '-dideoxynucleotide monomer moieties are provided.
　一実施形態において、３’末端にリン酸から誘導される基を有する第一のオリゴヌクレオチドの３’－末端のオリゴヌクレオチドモノマー部分は、天然型のリボヌクレオチド、天然型の２’－デオキシヌクレオチド、または２’－位水酸基がメチル化された２’－Ｏ－メチルヌクレオチド、あるいは２’－位にフルオロ基などの置換基が導入された２’－置換－２’－デオキシヌクレオチド誘導体である。 In one embodiment, the oligonucleotide monomer portion at the 3'-end of the first oligonucleotide having a group derived from phosphate at the 3'-end is a naturally occurring ribonucleotide, a naturally occurring 2'-deoxynucleotide, Alternatively, it is a 2'-O-methyl nucleotide in which the hydroxyl group at the 2'-position is methylated, or a 2'-substituted-2'-deoxynucleotide derivative in which a substituent such as a fluoro group is introduced at the 2'-position.
　一実施形態において、テンプレート核酸は、３’末端にリン酸から誘導される基を有する第一のオリゴヌクレオチドまたはその改変体および５’末端に５’－アミノ－５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体の両方のオリゴヌクレオチドまたはその改変体が連続して相補的な配列、あるいは安定な二本鎖を形成できる配列および鎖長を有するものである。また、テンプレート核酸は、第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体とは独立した核酸だけでなく、どちらか一方の断片、あるいは両方の断片と繋がったものも含む。 In one embodiment, the template nucleic acid has a first oligonucleotide or variant thereof having a phosphate-derived group at the 3' end and a 5'-amino-5'-deoxynucleotide monomer moiety at the 5' end. Both oligonucleotides of the second oligonucleotide or its variant or its variants have consecutive complementary sequences, or sequences and chain lengths that allow them to form a stable double strand. In addition, the template nucleic acid includes not only a nucleic acid independent of the first oligonucleotide or its variant and the second oligonucleotide or its variant, but also a nucleic acid linked to a fragment of either one or both fragments. .
　一実施形態において、各オリゴヌクレオチドまたはその改変体の鎖長は５～１０００塩基である。好ましくは５～２００塩基であり、あるいは１０～１００塩基であり得る。 In one embodiment, each oligonucleotide or variant thereof has a chain length of 5 to 1000 bases. It is preferably 5 to 200 bases, or may be 10 to 100 bases.
　一実施形態において、第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体の各オリゴヌクレオチドまたはその改変体は、それぞれ独立した配列でもよく、また１本に繋がったものでも良い。後者の場合、３’末端にリン酸から誘導される基を有し、５’末端に５’－アミノ－５’－デオキシヌクレオチドモノマー部分を有するオリゴヌクレオチドとなり、連結反応により環状のオリゴヌクレオチドを与える。 In one embodiment, each of the first oligonucleotide or its variant and the second oligonucleotide or its variant may have an independent sequence, or may be connected into one. . In the latter case, the oligonucleotide has a group derived from phosphoric acid at the 3' end and a 5'-amino-5'-deoxynucleotide monomer moiety at the 5' end, and a cyclic oligonucleotide is obtained by a ligation reaction. .
　一実施形態において、テンプレート核酸の鎖長は、連結させる第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体が連続して結合可能な配列を有しており、各オリゴヌクレオチドまたはその改変体に相補的な配列全てを含む、あるいは一部の配列を含むものである。またテンプレート核酸の配列は、各オリゴヌクレオチドまたはその改変体と完全に相補的な配列、あるいは二本鎖の形成を妨げない範囲のミスマッチ塩基対を含む配列でもよい。 In one embodiment, the chain length of the template nucleic acid is such that the first oligonucleotide or a variant thereof and the second oligonucleotide or variant thereof to be linked have a sequence that allows for continuous binding, and each oligonucleotide or a variant thereof, including all or a part of the sequence complementary to the variant thereof. Furthermore, the sequence of the template nucleic acid may be a sequence that is completely complementary to each oligonucleotide or a variant thereof, or a sequence that contains mismatched base pairs within a range that does not prevent the formation of double strands.
　一実施形態において、本開示において用いられる連結反応は、第一のオリゴヌクレオチドまたはその改変体の3’末端にチオリン酸基を有しており、これを１－フルオロ－２，４－ジニトロベンゼン（ＤＮＦＢ）等の活性化剤により活性化して第二のオリゴヌクレオチドまたはその改変体と反応させ、ホスホロアミデート結合を形成させることで２つのオリゴヌクレオチドまたはその改変体を結合させるオリゴヌクレオチドの連結反応を含む。活性化剤はチオリン酸の硫黄原子と反応してリン酸基を活性化できる化合物であれば、１－フルオロ－２，４－ジニトロベンゼン（ＤＮＦＢ）に限定されない。本開示の活性剤を用いることで、副反応が起こらず、収率よく連結反応を行うことが可能となる。 In one embodiment, the ligation reaction used in this disclosure has a thiophosphate group at the 3' end of the first oligonucleotide or variant thereof, which is combined with 1-fluoro-2,4-dinitrobenzene ( A ligation reaction of oligonucleotides that binds two oligonucleotides or their variants by activation with an activator such as DNFB) and reacting with a second oligonucleotide or its variant to form a phosphoroamidate bond. including. The activator is not limited to 1-fluoro-2,4-dinitrobenzene (DNFB) as long as it is a compound that can activate the phosphoric acid group by reacting with the sulfur atom of thiophosphoric acid. By using the activator of the present disclosure, side reactions do not occur and it becomes possible to perform the linking reaction with good yield.
　一実施形態において、本開示において用いられる連結反応は、第一のオリゴヌクレオチドまたはその改変体の３’末端にリン酸基またはチオリン酸基を有しており、これをカルボジイミド系の活性化剤（ＥＤＣ、ＤＣＣ等）により活性化して第二のオリゴヌクレオチドまたはその改変体と反応させ、ホスホロアミデート結合を形成させることで２つのオリゴヌクレオチドまたはその改変体を結合させるオリゴヌクレオチドの連結反応を含む。 In one embodiment, the ligation reaction used in the present disclosure has a phosphate group or thiophosphate group at the 3' end of the first oligonucleotide or variant thereof, which is combined with a carbodiimide-based activator ( (EDC, DCC, etc.) and react with a second oligonucleotide or a variant thereof to form a phosphoroamidate bond, thereby linking the oligonucleotides to link two oligonucleotides or variants thereof. .
　一実施形態において、本開示において用いられる連結反応は、３’末端にリン酸から誘導される基を導入した第一のオリゴヌクレオチドまたはその改変体を、あらかじめ活性化剤により処理して活性な中間体（活性化第一のオリゴヌクレオチドまたはその改変体）に変換した後に、５’末端に５’－アミノ－２’－フルオロ－２’，５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体とテンプレート核酸の混合物に加えて行われる。 In one embodiment, the ligation reaction used in the present disclosure is performed by treating a first oligonucleotide or a variant thereof having a phosphoric acid-derived group introduced at its 3' end with an activating agent to activate an active intermediate. a second oligonucleotide having a 5'-amino-2'-fluoro-2',5'-deoxynucleotide monomer moiety at its 5' end after conversion to an activated first oligonucleotide or a variant thereof; or in addition to a mixture of the variant and the template nucleic acid.
　一実施形態において、本開示において用いられる連結反応は、３’末端にリン酸から誘導される基を導入した第一のオリゴヌクレオチドまたはその改変体と、５’末端に５’－アミノ－２’－フルオロ－２’，５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体、およびテンプレート核酸の３種を混合させたのちに、活性化剤を加えて行われる。 In one embodiment, the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end. - After mixing the second oligonucleotide having a fluoro-2',5'-deoxynucleotide monomer moiety or a variant thereof, and a template nucleic acid, an activating agent is added.
　一実施形態において、本開示において用いられる連結反応は、３’末端にリン酸から誘導される基を導入した第一のオリゴヌクレオチドまたはその改変体と、５’末端に５’－アミノ－２’－フルオロ－２’，５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体、およびテンプレート核酸の３種のオリゴヌクレオチドが、いずれもＲＮＡ配列およびその誘導体である連結反応を含む。 In one embodiment, the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end. - A second oligonucleotide having a fluoro-2',5'-deoxynucleotide monomer moiety or a variant thereof, and a template nucleic acid, the three oligonucleotides comprising a ligation reaction, all of which are RNA sequences and derivatives thereof.
　一実施形態において、本開示において用いられる連結反応は、３’末端にリン酸から誘導される基を導入した第一のオリゴヌクレオチドまたはその改変体と、５’末端に５’－アミノ－２’－フルオロ－２’，５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体が共にＲＮＡ配列およびその誘導体であり、テンプレート核酸が、ＤＮＡ配列およびその誘導体である連結反応を含む。 In one embodiment, the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end. - A ligation reaction in which the second oligonucleotide or variant thereof having a fluoro-2',5'-deoxynucleotide monomer moiety is both an RNA sequence and a derivative thereof, and the template nucleic acid is a DNA sequence and a derivative thereof.
　一実施形態において、本開示において用いられる連結反応は、３’末端にリン酸から誘導される基を導入した第一のオリゴヌクレオチドまたはその改変体と、５’末端に５’－アミノ－２’－フルオロ－２’，５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体、およびテンプレート核酸の３種のオリゴヌクレオチドが、いずれもＤＮＡ配列およびその誘導体である連結反応を含む。 In one embodiment, the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end. - A second oligonucleotide or a variant thereof having a fluoro-2',5'-deoxynucleotide monomer moiety, and a template nucleic acid, the three oligonucleotides comprising a ligation reaction, all of which are DNA sequences and derivatives thereof.
　一実施形態において、本開示において用いられる連結反応は、３’末端にリン酸から誘導される基を導入した第一のオリゴヌクレオチドまたはその改変体と、５’末端に５’－アミノ－２’－フルオロ－２’，５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体が共にＤＮＡ配列およびその誘導体であり、テンプレート核酸が、ＲＮＡ配列およびその誘導体である連結反応を含む。 In one embodiment, the ligation reaction used in the present disclosure involves using a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at its 3' end, and a 5'-amino-2' oligonucleotide at its 5' end. - A ligation reaction in which the second oligonucleotide or variant thereof having a fluoro-2',5'-deoxynucleotide monomer moiety is both a DNA sequence and a derivative thereof, and the template nucleic acid is an RNA sequence and a derivative thereof.
　一実施形態において、本開示において用いられる連結反応は、３’末端にリン酸から誘導される基を導入した第一のオリゴヌクレオチドまたはその改変体、５’末端に５’－アミノ－２’－フルオロ－２’，５’－デオキシヌクレオチドモノマー部分を有する第二のオリゴヌクレオチドまたはその改変体、およびテンプレート核酸の全て、あるいは一部の配列が、ＲＮＡ配列およびその誘導体とＤＮＡ配列およびその誘導体からなる複合配列である連結反応を含む。
　（製造に使うツール、試薬） In one embodiment, the ligation reaction used in the present disclosure includes a first oligonucleotide or a variant thereof having a phosphate-derived group introduced at the 3' end, 5'-amino-2'- The second oligonucleotide or its variant having a fluoro-2',5'-deoxynucleotide monomer moiety, and all or part of the template nucleic acid sequence consist of an RNA sequence and its derivatives and a DNA sequence and its derivatives. Contains ligation reactions that are complex sequences.
(Tools and reagents used in manufacturing)
　別の局面において、本開示は、本明細書に記載される第一のオリゴヌクレオチドまたはその改変体または第二のオリゴヌクレオチドまたはその改変体を含む、それぞれ第二のオリゴヌクレオチドまたはその改変体または第一のオリゴヌクレオチドまたはその改変体を用いて連結オリゴヌクレオチドまたはその改変体を製造するための組成物を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）に記載される任意の形態を適宜組み合わせて適用することができることが理解される。 In another aspect, the present disclosure provides a first oligonucleotide or a variant thereof or a second oligonucleotide or a variant thereof, respectively, as described herein. A composition for producing a linked oligonucleotide or a variant thereof using one oligonucleotide or a variant thereof is provided. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
　1つの実施形態では、本明細書に記載される第一のオリゴヌクレオチドまたはその改変体を含む、第二のオリゴヌクレオチドまたはその改変体を用いて連結オリゴヌクレオチドまたはその改変体を製造するための組成物を提供する。 In one embodiment, a composition for producing a linked oligonucleotide or variant thereof using a second oligonucleotide or variant thereof comprising a first oligonucleotide or variant thereof described herein. provide something.
　1つの実施形態では、本明細書に記載される第二のオリゴヌクレオチドまたはその改変体を含む、第一のオリゴヌクレオチドまたはその改変体を用いて連結オリゴヌクレオチドまたはその改変体を製造するための組成物を提供する。 In one embodiment, a composition for producing a linked oligonucleotide or variant thereof using a first oligonucleotide or variant thereof, comprising a second oligonucleotide or variant thereof as described herein. provide something.
　1つの実施形態では、連結オリゴヌクレオチドまたはその改変体を製造するための組成物はアミダイド体のまたはデオキシヌクレオシド、または３’位が水酸基であるまたはデオキシヌクレオシドを含む。 In one embodiment, the composition for producing a linked oligonucleotide or a variant thereof comprises an amidite or deoxynucleoside, or a deoxynucleoside in which the 3' position is a hydroxyl group.
　他の局面において、本開示は第一のオリゴヌクレオチドまたはその改変体もしくは第二のオリゴヌクレオチドまたはその改変体、またはそれらの組み合わせを含む、テンプレート核酸を用いて連結オリゴヌクレオチドまたはその改変体を製造するための組成物を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）に記載される任意の形態を適宜組み合わせて適用することができることが理解される。 In other aspects, the present disclosure provides for producing linked oligonucleotides or variants thereof using template nucleic acids, including a first oligonucleotide or a variant thereof or a second oligonucleotide or a variant thereof, or a combination thereof. Provides a composition for. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
　別の局面において、本開示はオリゴヌクレオチドまたはその改変体を連結するためのキットであって、第一のオリゴヌクレオチドまたはその改変体と、第二のオリゴヌクレオチドまたはその改変体と、活性化剤と、テンプレート核酸とを含むキットを提供する。 In another aspect, the present disclosure is a kit for linking oligonucleotides or variants thereof, comprising a first oligonucleotide or variant thereof, a second oligonucleotide or variant thereof, and an activator. and a template nucleic acid.
　一実施形態において、本開示は第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体を含む、連結オリゴヌクレオチドまたはその改変体を製造するための原料組成物を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）に記載される任意の形態を適宜組み合わせて適用することができることが理解される。 In one embodiment, the present disclosure provides a raw material composition for producing a linked oligonucleotide or a variant thereof, including a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
　一局面において、本開示は前記第二のオリゴヌクレオチドまたはその改変体を製造するための原料が、２’位にフルオロ基、５’位にアミノ基を有するヌクレオシドを含む、原料組成物を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）に記載される任意の形態を適宜組み合わせて適用することができることが理解される。
　ここでは、原料として、例えば、ヌクレオシド、モノマーまたは置換基導入試薬などが例示される。 In one aspect, the present disclosure provides a raw material composition, wherein the raw material for producing the second oligonucleotide or a variant thereof includes a nucleoside having a fluoro group at the 2' position and an amino group at the 5' position. . It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
Here, examples of raw materials include nucleosides, monomers, and substituent-introducing reagents.
　別の局面において、本開示は第一／第二のオリゴヌクレオチドまたはその改変体の原料（モノマーまたは置換基導入試薬等）を含む、第一のオリゴヌクレオチドまたはその改変体、および／または第二のオリゴヌクレオチドまたはその改変体を製造するための組成物を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）に記載される任意の形態を適宜組み合わせて適用することができることが理解される。 In another aspect, the present disclosure provides a first oligonucleotide or a variant thereof, including a raw material (a monomer or a substituent introduction reagent, etc.) for the first/second oligonucleotide or a variant thereof, and/or a second oligonucleotide or a variant thereof. Compositions for producing oligonucleotides or variants thereof are provided. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
　一実施形態において、第一／第二のオリゴヌクレオチドまたはその改変体の原料が、アミダイド体のヌクレオシド、または３’位が水酸基であるヌクレオシドである。 In one embodiment, the raw material for the first/second oligonucleotide or a variant thereof is an amidite nucleoside or a nucleoside having a hydroxyl group at the 3' position.
　別の局面において、本開示はＡ）合成を所望する核酸配列の情報を得る工程、Ｂ）Ａ）の情報に基づき、合成したい配列の一部を複数合成する工程であって、ここで、各配列は、第一および第二のオリゴヌクレオチドまたはその改変体の特徴を有する、工程、ならびにＣ）必要に応じて連結反応に必要な試薬の存在下で、連結条件に配置する工程を包含する、所望の核酸配列を有するオリゴヌクレオチドまたはその改変体の製造方法を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）に記載される任意の形態を適宜組み合わせて適用することができることが理解される。 In another aspect, the present disclosure provides a step of A) obtaining information on a nucleic acid sequence desired to be synthesized; B) synthesizing a plurality of parts of the sequence desired to be synthesized based on the information in A), wherein each the sequence has characteristics of the first and second oligonucleotides or variants thereof, and C) placing them in ligation conditions, optionally in the presence of reagents necessary for the ligation reaction. A method for producing an oligonucleotide having a desired nucleic acid sequence or a variant thereof is provided. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
　別の局面において、本開示は、Ａ）合成を所望する核酸配列の情報を得る工程、Ｂ）Ａ）の情報に基づき、合成したい配列の一部を複数合成する工程であって、ここで、各配列は、第一および第二のオリゴヌクレオチドまたはその改変体の特徴を有する、工程、ならびにＣ）必要に応じて連結反応に必要な試薬と、Ｂ）で合成された部分配列を含む核酸とをパッケージとする工程を包含する、所望のオリゴヌクレオチドまたはその改変体の製造キットの製造方法を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）に記載される任意の形態を適宜組み合わせて適用することができることが理解される。 In another aspect, the present disclosure provides a step of A) obtaining information on a nucleic acid sequence desired to be synthesized, B) synthesizing a plurality of parts of the sequence desired to be synthesized based on the information in A), wherein: each sequence has the characteristics of the first and second oligonucleotides or variants thereof; and C) optionally the reagents necessary for the ligation reaction; and B) a nucleic acid comprising the partial sequence synthesized in step B). Provided is a method for producing a kit for producing a desired oligonucleotide or a variant thereof, which includes the step of packaging the oligonucleotide. It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof).
　一実施形態において、Ａ）合成を所望する核酸配列の情報を得る工程は、シーケンス解析等を行って、核酸の配列情報を読み取る工程を含む。 In one embodiment, A) the step of obtaining information on the nucleic acid sequence desired to be synthesized includes the step of performing sequence analysis or the like to read the sequence information of the nucleic acid.
　一実施形態において、連結反応に必要な試薬が、テンプレート核酸、活性化剤を含む。
　（新規オリゴヌクレオチドまたはその改変体） In one embodiment, the reagents necessary for the ligation reaction include a template nucleic acid, an activator.
(Novel oligonucleotide or variant thereof)
　一局面において、本開示は以下の式

（式中、Ｙは、水素基またはアルコキシ基であり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ｘは、ヒドロキシ基、またはハロゲンであり、Ａは、各出現において独立して核酸、または核酸誘導体であり、ｎは１以上の整数であり、ｐは１以上の整数であり、Ｒ１は５’－位が水酸基である核酸、または核酸誘導体、Ｒ２は３’－位が水酸基またはリン酸基またはチオリン酸基である核酸、または核酸誘導体であるか、Ｒ１およびＲ２は、一緒になって単結合を形成してもよく、Ｒ３が置換されていてもよいアミノ基である）で示される化合物を提供する。ここで記載される各種実施形態は、（オリゴヌクレオチドまたはその改変体の連結）等に記載される任意の形態を適宜組み合わせて適用することができることが理解される。理論に束縛されることを望まないが、ヌクレアーゼ耐性が高いオリゴヌクレオチドまたはその改変体を達成することができる。 In one aspect, this disclosure provides the following formula:

(In the formula, Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine , 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine. , X is a hydroxy group or a halogen, A is independently at each occurrence a nucleic acid or a nucleic acid derivative, n is an integer of 1 or more, p is an integer of 1 or more, R 1 is a nucleic acid or a nucleic acid derivative in which the 5'-position is a hydroxyl group, R 2 is a nucleic acid or a nucleic acid derivative in which the 3'-position is a hydroxyl group, a phosphoric acid group, or a thiophosphoric acid group; R 1 and R 2 are may be taken together to form a single bond, and R 3 is an optionally substituted amino group). It is understood that the various embodiments described herein can be applied by appropriately combining any forms described in (Linking of oligonucleotides or variants thereof) and the like. Without wishing to be bound by theory, oligonucleotides or variants thereof that are highly resistant to nucleases can be achieved.
　一実施形態において、Ｙは、メトキシ基であり、前記Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、前記Ｘは、フルオロ基であり、Ｒ１は水素であり、Ｒ２はヒドロキシ基である。 In one embodiment, Y is a methoxy group, and each B is independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , said X is a fluoro group, R 1 is hydrogen, and R 2 is a hydroxy group.
　一実施形態において、Ｙは、メトキシ基であり、前記Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、前記Ｘは、フルオロ基であり、Ｒ１およびＲ２は、一緒になって単結合を形成している。 In one embodiment, Y is a methoxy group, and each B is independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , the X is a fluoro group, and R 1 and R 2 together form a single bond.
　一実施形態において、Ｙは、メトキシ基である。 In one embodiment, Y is a methoxy group.
　一実施形態において、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、好ましくはアデニン、シトシン、メチルシトシン、グアニン、チミン、またはウラシルである。 In one embodiment, B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1 - methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine, preferably adenine, cytosine, methylcytosine , guanine, thymine, or uracil.
　一実施形態において、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、またはウラシルである。 In one embodiment, B is each independently adenine, cytosine, methylcytosine, guanine, thymine, or uracil.
　一実施形態において、ｎは５から１０００の整数である。好ましくはｎは５から９００の整数、ｎは５から８００の整数、ｎは５から７００の整数、ｎは５から６００の整数、ｎは５から５００の整数、ｎは５から４００の整数、ｎは５から３００の整数、ｎは５から２００の整数、ｎは５から１００の整数であり、より好ましくは１０から１００の整数である。 In one embodiment, n is an integer from 5 to 1000. Preferably n is an integer from 5 to 900, n is an integer from 5 to 800, n is an integer from 5 to 700, n is an integer from 5 to 600, n is an integer from 5 to 500, n is an integer from 5 to 400, n is an integer from 5 to 300, n is an integer from 5 to 200, n is an integer from 5 to 100, and more preferably an integer from 10 to 100.
　一実施形態において、ｐは５から１０００の整数である。好ましくはｐは５から９００の整数、ｐは５から８００の整数、ｐは５から７００の整数、ｐは５から６００の整数、ｐは５から５００の整数、ｐは５から４００の整数、ｐは５から３００の整数、ｐは５から２００の整数、ｐは５から１００の整数であり、より好ましくは１０から１００の整数である。 In one embodiment, p is an integer from 5 to 1000. Preferably p is an integer from 5 to 900, p is an integer from 5 to 800, p is an integer from 5 to 700, p is an integer from 5 to 600, p is an integer from 5 to 500, p is an integer from 5 to 400, p is an integer from 5 to 300, p is an integer from 5 to 200, p is an integer from 5 to 100, and more preferably an integer from 10 to 100.
　一実施形態において、Ｘは、ヒドロキシ基、またはハロゲンであり、好ましくはXはハロゲンであり、より好ましくはフルオロ基である。Xがフルオロ基であるオリゴヌクレオチドまたはその改変体は、核酸分解酵素に耐性を有する（分解され難い）、二本鎖形成を安定化する等の利点を有する。 In one embodiment, X is a hydroxy group or a halogen, preferably a halogen, more preferably a fluoro group. Oligonucleotides in which X is a fluoro group or variants thereof have advantages such as being resistant to nucleases (hard to be degraded) and stabilizing double-strand formation.
　一実施形態において、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、好ましくは、Ａは核酸である。より好ましくはＡはＤＮＡまたはＲＮＡである。ここで核酸誘導体はホスホロチオエート核酸、ＬＮＡ修飾核酸、２’－ＯＭｅ修飾核酸、２’－Ｆ修飾核酸、２’－ＭＯＥ修飾核酸、ＰＭＯ核酸、またはＰＮＡ核酸である。 In one embodiment, A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, preferably A is a nucleic acid. More preferably A is DNA or RNA. Here, the nucleic acid derivative is a phosphorothioate nucleic acid, an LNA modified nucleic acid, a 2'-OMe modified nucleic acid, a 2'-F modified nucleic acid, a 2'-MOE modified nucleic acid, a PMO nucleic acid, or a PNA nucleic acid.
　一実施形態において、Ｒ１は５’－位が水酸基である核酸、または核酸誘導体、Ｒ２は３’－位が水酸基またはリン酸基またはチオリン酸基である核酸、または核酸誘導体であるか、Ｒ１およびＲ２は、一緒になって単結合を形成してもよい。 In one embodiment, R 1 is a nucleic acid or a nucleic acid derivative whose 5'-position is a hydroxyl group, R 2 is a nucleic acid or a nucleic acid derivative whose 3'-position is a hydroxyl group, a phosphate group, or a thiophosphoric acid group, or R 1 and R 2 may be taken together to form a single bond.
　一実施形態において、Ｒ３は置換されていてもよいアミノ基である。 In one embodiment, R 3 is an optionally substituted amino group.
　一実施形態において、Ｒ３は置換されていないアミノ基である。 In one embodiment, R 3 is an unsubstituted amino group.
　一実施形態において、Ｒ３はアミノ基はＣ１－６アルキル基によって置換されており、好ましくは、メチル基またはエチル基によって置換されている。 In one embodiment, the amino group of R 3 is substituted by a C 1-6 alkyl group, preferably by a methyl group or an ethyl group.
　一実施形態において、本開示は第一のオリゴヌクレオチドまたはその改変体の５’末端と第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体の複数と、前記第一のオリゴヌクレオチドまたはその改変体と、前記第二のオリゴヌクレオチドまたはその改変体は、連結されている化合物を含む。 In one embodiment, the present disclosure provides a plurality of oligonucleotides or variants thereof in which the 5' end of the first oligonucleotide or variant thereof is linked to the 3' end of the second oligonucleotide or variant thereof; The first oligonucleotide or its variant and the second oligonucleotide or its variant include a linked compound.
　一実施形態において、本開示はＲ１およびＲ２が、一緒になって単結合を形成しており、それにより、環状オリゴヌクレオチドまたはその改変体が形成している化合物を含む。 In one embodiment, the present disclosure includes compounds in which R 1 and R 2 are taken together to form a single bond, thereby forming a cyclic oligonucleotide or variant thereof.
　一実施形態において、本開示は、第一のオリゴヌクレオチドまたはその改変体の５’末端に、テンプレート核酸が結合している化合物を含む。 In one embodiment, the present disclosure includes a compound in which a template nucleic acid is attached to the 5' end of a first oligonucleotide or a variant thereof.
　一実施形態において、本開示は、第二のオリゴヌクレオチドまたはその改変体の３’末端に、テンプレート核酸が結合している化合物を含む。 In one embodiment, the present disclosure includes a compound in which a template nucleic acid is attached to the 3' end of a second oligonucleotide or a variant thereof.
　一実施形態において、本開示は、第一のオリゴヌクレオチドまたはその改変体の５’末端と第二のオリゴヌクレオチドまたはその改変体の３’末端とが、テンプレート核酸を介して結合している化合物を含む。 In one embodiment, the present disclosure provides a compound in which the 5' end of a first oligonucleotide or a variant thereof and the 3' end of a second oligonucleotide or variant thereof are linked via a template nucleic acid. include.
　（医薬他応用）
　本開示は、医薬として応用することができる。
　例えば、本開示の組成物および方法は、それを必要とする個体を処置するために利用することができる。特定の実施形態では、個体は、哺乳動物、例えばヒトなど、または非ヒト哺乳動物である。動物、例えばヒトなどに投与された場合、組成物または化合物は、好ましくは、例えば、本開示の化合物と、薬学的に許容される担体とを含む医薬組成物として投与される。薬学的に許容される担体は、当技術分野で周知であり、例えば、水溶液、例えば、水もしくは緩衝生理食塩水など、または他の溶媒もしくはビヒクル、例えば、グリコール、グリセロール、油、例えば、オリーブ油など、または注射用の有機エステルが挙げられる。好ましい実施形態では、このような医薬組成物がヒトへの投与、特に侵襲経路の投与のためである場合（すなわち、例えば、上皮バリアを介した輸送または拡散を回避する注射またはインプランテーションなどの経路）、水溶液はパイロジェンを含まない、またはパイロジェンを実質的に含まない。賦形剤は、例えば、剤の遅延放出を実行するように、または１つもしくは複数の細胞、組織または器官を選択的にターゲットとするよう選択することができる。医薬組成物は、単位剤形、例えば、錠剤、カプセル剤（スプリンクルカプセル剤およびゼラチンカプセル剤を含む）、顆粒剤、再構成用に凍結乾燥されたもの、散剤、液剤、シロップ剤、坐剤、または注射などであり得る。組成物はまた、経皮的送達システム、例えば、皮膚パッチ中に存在することもできる。組成物はまた、局所的投与に対して適切な液剤、例えば点眼剤などの中に存在することもできる。 (Pharmaceutical and other applications)
The present disclosure can be applied as a medicine.
For example, the compositions and methods of this disclosure can be utilized to treat individuals in need thereof. In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, etc. , or injectable organic esters. In a preferred embodiment, such pharmaceutical compositions are for administration to humans, particularly by invasive routes (i.e., routes such as injection or implantation that avoid transport or diffusion across epithelial barriers). ), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to effect delayed release of the agent or to selectively target one or more cells, tissues or organs. The pharmaceutical compositions may be prepared in unit dosage forms, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilized for reconstitution, powders, solutions, syrups, suppositories, Or it could be an injection. The composition can also be presented in a transdermal delivery system, such as a skin patch. The compositions can also be presented in solutions suitable for topical administration, such as eye drops.
　本開示はまた、ゲノム編集技術において広く利用することができる。限定するものではないが、例えば、ゲノム編集で用いられているｓｇＲＮＡに応用することができる（J.　Filippova　et　al.，　Biochimie　167　2019　49-60、Ayal　Hendel　et　al.　　nature　biotechnology　33　2015　985-989）。理論に束縛されることを望まないが、本開示の改変（修飾）塩基を含むオリゴヌクレオチドまたはその改変体（オリゴ核酸）を用いることにより活性が上がるようであることから、本開示の技術を用いて合成した修飾塩基を含むオリゴ核酸のフラグメントをつなげて作製することが応用として想定され得る。別の実施形態としては、T.　Seya　et　al.　Advanced　Drug　Delivery　Reviews　147　2019　37-43に掲載されているような比較的長めの２本鎖を、テンプレートのＤＮＡを用いずに、お互いの鎖がテンプレートになるように作製することも想定され得る（図１１）。 The present disclosure can also be widely used in genome editing technology. For example, but not limited to, it can be applied to sgRNA used in genome editing (J. Filippova et al., Biochimie 167 2019 49-60, Ayal Hendel et al. Nature Biotechnology 33 2015 9 85-989 ). Without wishing to be bound by theory, it appears that the use of oligonucleotides or modified versions thereof (oligonucleic acids) containing the modified (modified) bases of the present disclosure increases activity. One possible application would be to connect fragments of oligonucleic acids containing modified bases synthesized by the method. Another embodiment is to create a relatively long double strand, as described in T. Seya et al. It may also be envisaged to produce it as a template (FIG. 11).
　別の実施形態としては、例えば、長鎖非翻訳ＲＮＡ（ｌｏｎｇ　ｎｏｎ－ｃｏｄｉｎｇ　ＲＮＡ；　ｌｎｃＲＮＡ）やｍＲＮＡについても対象とされ得る。 In another embodiment, for example, long non-coding RNA (lncRNA) and mRNA may also be targeted.
　投与が考えられる「対象」は、ヒト（すなわち、いずれかの年齢群の男性または女性、例えば、小児科の対象（例えば、幼児、児童、思春期）または成人の対象（例えば、若年成人、中年成人、または高齢成人））および／または他の非ヒト動物、例えば哺乳動物（例えば霊長類（例えばカニクイザル、アカゲザル）、商業的に関係する哺乳動物、例えば牛、ブタ、馬、羊、ヤギ、猫、および／または犬）および鳥類（例えば、商業的に関係する鳥類、例えば鶏、鴨類、ガチョウ、および／または七面鳥）、爬虫類、両生類、および魚類を含むが、それらに限定されない。ある実施形態において、非ヒト動物は哺乳動物である。非ヒト動物は発生のいずれかの時期にある雄または雌であり得る。非ヒト動物はトランスジェニック動物であり得る。 "Subjects" to which administration is contemplated include humans (i.e., men or women of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults). adults (or older adults)) and/or other non-human animals, such as mammals, such as primates (e.g. cynomolgus macaques, rhesus macaques), commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats. , and/or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and/or turkeys), reptiles, amphibians, and fish. In certain embodiments, the non-human animal is a mammal. The non-human animal can be male or female at any stage of development. A non-human animal can be a transgenic animal.
　本明細書において用いられる場合、特に限定して言及しない限り、用語「治療する」、「治療すること」、および「治療」（英語では、ｔｒｅａｔ、ｔｒｅａｔｉｎｇ、ｔｒｅａｔｍｅｎｔ）は、対象が疾患、障害または状態に罹患している間に起こり、疾患、障害または状態の深刻さを減少させるかまたは疾患、障害または状態の進行を遅延もしくは減速させる行為（狭義の「治療」（ｔｈｅｒａｐｙ））であるか、対象が障害に罹患し始める前において、疾患、障害または状態になるのを阻害するかまたは実際に疾患、障害または状態になってしまった場合の深刻さを減少させる行為（「予防」）もまた包含される。 As used herein, unless specifically stated otherwise, the terms "treat," "treating," and "treatment" (in English, treat, treating, treatment) refer to a disease, disorder, or is an act that occurs while suffering from a condition and that reduces the severity of the disease, disorder or condition or delays or slows the progression of the disease, disorder or condition (in the narrow sense of "therapy"); The act of preventing a disease, disorder, or condition from developing or reducing the severity of a disease, disorder, or condition if the subject actually develops the disorder (“prevention”) Included.
　一般的に、化合物の「有効量」は所望の生物学的応答を惹起し、例えば、疾患、障害または状態を治療するのに十分な量を指す。当業者には当然のことながら、本開示の化合物の有効量は、所望の生物学的エンドポイント、化合物の薬物動態、治療されようとする疾患、障害または状態、投与の様式、ならびに年齢、健康、および対象などの因子に依存して変わり得る。有効量は治療および予防の治療を包含する。 Generally, an "effective amount" of a compound refers to an amount sufficient to elicit a desired biological response, eg, treat a disease, disorder, or condition. Those skilled in the art will appreciate that an effective amount of a compound of the present disclosure will depend on the desired biological endpoint, the pharmacokinetics of the compound, the disease, disorder or condition being treated, the mode of administration, and the age, health, etc. , and may vary depending on factors such as the subject. Effective amounts include therapeutic and prophylactic treatments.
　本明細書において用いられる場合、特に限定して言及しない限り、化合物の「治療有効量」は、疾患、障害または状態の治療に際して治療効果を提供するか、または疾患、障害または状態と結びついた１つまたは２つ以上の症状を遅延させるもしくは最小化するのに十分な量を指す。化合物の治療有効量は、単独でまたは他の療法との組み合わせで、疾患、障害または状態の治療に際して治療効果を提供する治療剤の量を意味する。用語「治療有効量」は、全般的な療法を改善する、疾患、障害または状態の症状もしくは原因を減少させるもしくは防ぐ、または別の治療剤の治療有効性を増強する量を包含し得る。 As used herein, unless specifically stated otherwise, a "therapeutically effective amount" of a compound means a "therapeutically effective amount" of a compound that provides a therapeutic effect in the treatment of a disease, disorder or condition or that is associated with the disease, disorder or condition. Refers to an amount sufficient to delay or minimize one or more symptoms. A therapeutically effective amount of a compound means that amount of the therapeutic agent, alone or in combination with other therapies, that provides a therapeutic effect in treating the disease, disorder, or condition. The term "therapeutically effective amount" can include an amount that improves overall therapy, reduces or prevents a symptom or cause of a disease, disorder or condition, or enhances the therapeutic effectiveness of another therapeutic agent.
　本明細書において用いられる場合、特に限定して言及しない限り、化合物の「予防有効量」は、疾患、障害または状態または疾患、障害または状態と結びついた１つもしくは２つ以上の症状を予防、あるいはその再発を予防するのに十分な量である。化合物の予防有効量は、単独でまたは他の剤との組み合わせで、疾患、障害または状態の予防に際して予防効果を提供する治療剤の量を意味する。用語「予防有効量」は、全般的な予防を改善するまたは別の予防剤の予防有効性を増強する量を包含し得る。 As used herein, unless specifically stated otherwise, a "prophylactically effective amount" of a compound means to prevent, prevent, or prevent a disease, disorder, or condition or one or more symptoms associated with the disease, disorder, or condition. or in an amount sufficient to prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent that, alone or in combination with other agents, provides a prophylactic effect in preventing the disease, disorder, or condition. The term "prophylactically effective amount" can include an amount that improves overall prophylaxis or enhances the prophylactic effectiveness of another prophylactic agent.
　例示的な疾患、障害または状態は、増殖性、神経系、免疫系、内分泌系、心血管系、血液系、炎症性、および細胞死によって特徴づけられる障害を含むが、それらに限定されない。 Exemplary diseases, disorders or conditions include, but are not limited to, proliferative, neurological, immune, endocrine, cardiovascular, hematological, inflammatory, and disorders characterized by cell death.
　本明細書において用いられる場合、増殖性の疾患、障害または状態は、癌、造血系の腫瘍、増殖性乳部疾患、肺の増殖性障害、結腸の増殖性障害、肝臓の増殖性障害、および卵巣の増殖性障害を含むが、それらに限定されない。 As used herein, proliferative diseases, disorders or conditions include cancer, tumors of the hematopoietic system, proliferative breast diseases, proliferative disorders of the lung, proliferative disorders of the colon, proliferative disorders of the liver, and including, but not limited to, ovarian proliferative disorders.
　本明細書に記載のオリゴヌクレオチドまたはその改変体は核酸医薬に用いることができる。
　（合成例） The oligonucleotides or variants thereof described herein can be used in nucleic acid medicines.
(Synthesis example)
　（本開示の化合物の製造法）
　以下に、本開示の化合物の製造法について、実施例に具体的な合成例を挙げているが、これに限定されるものではなく、以下に代表的なスキームを挙げて説明するが、本開示はもとよりこれらに限定されるものではない。 (Method for producing the compound of the present disclosure)
Below, specific synthesis examples are given in Examples regarding the method for producing the compound of the present disclosure, but the present disclosure is not limited thereto. Of course, it is not limited to these.
　本開示の化合物は、これらに限定されないが、例えば、下記に記した製造法によって製造することができる。これらの製造法は、有機合成化学を習熟している者の知識に基づき、適宜改良することができる。下記製造法において、原料として用いられる化合物は、反応に支障をきたさない限り、それらの塩を用いてもよい。
　論文記載（Ueno,　Y.,　Hirai,　M.,　Yoshikawa,　K.,　Kitamura,　Y.,　Hirata,　Y.,　Kiuchi,　K.,　Kitade,　Y.　Tetrahedron,　2008,　64,　11328-11334.、Hideto　Maruyama　et　al.,　Nucleic　Acids　Research,　2017,　Vol.　45,　No.　12，7042-7048）の方法を参考にして、５’位にアミノ基、および２’位にハロまたは－ＯＨが置換されているヌクレオチドまたはその改変体のアミダイド化合物を合成する。この化合物をオリゴヌクレオチドまたはその改変体と反応させ、５’末端に５’位にアミノ基、および２’位にハロまたは－ＯＨが置換されているヌクレオチド末端を有する第二のオリゴヌクレオチドまたはその改変体を合成する。第一のヌクレオチドまたはその改変体を活性剤と反応させ、活性化第一のオリゴヌクレオチドまたはその改変体を合成する。活性化第一のオリゴヌクレオチドまたはその改変体を第二のオリゴヌクレオチドまたはその改変体と必要に応じてテンプレート核酸の存在下で反応させて、第一のオリゴヌクレオチドまたはその改変体と第二のオリゴヌクレオチドまたはその改変体を連結し、オリゴヌクレオチドまたはその改変体を生じさせることができる。 The compounds of the present disclosure can be produced, for example, by the production methods described below, although they are not limited thereto. These production methods can be modified as appropriate based on the knowledge of those skilled in organic synthetic chemistry. In the following production method, salts of the compounds used as raw materials may be used as long as they do not interfere with the reaction.
Paper description (Ueno, Y., Hirai, M., Yoshikawa, K., Kitamura, Y., Hirata, Y., Kiuchi, K., Kitade, Y. Tetrahedron, 2008, 64, 11328-11334., Hideto Maruyama et al., Nucleic Acids Research, 2017, Vol. 45, No. 12, 7042-7048), an amino group is substituted at the 5' position and a halo or -OH is substituted at the 2' position. Synthesize amidide compounds of nucleotides or their variants. This compound is reacted with an oligonucleotide or a variant thereof, and a second oligonucleotide or a variant thereof having a nucleotide terminus in which an amino group is substituted at the 5' position and a halo or -OH is substituted at the 2' position is obtained. Synthesize the body. The first nucleotide or variant thereof is reacted with an activating agent to synthesize an activated first oligonucleotide or variant thereof. The activated first oligonucleotide or its variant is reacted with a second oligonucleotide or its variant, optionally in the presence of a template nucleic acid, to form the first oligonucleotide or its variant and the second oligonucleotide. Nucleotides or variants thereof can be linked to generate oligonucleotides or variants thereof.
　以上、本開示を、理解の容易のために好ましい実施形態を示して説明してきた。以下に、実施例に基づいて本開示を説明するが、上述の説明および以下の実施例は、例示の目的のみに提供され、本開示を限定する目的で提供したのではない。従って、本開示の範囲は、本明細書に具体的に記載された実施形態にも実施例にも限定されず、特許請求の範囲によってのみ限定される。 The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.
　本実施例では、本開示の化合物の製造、用途に関する実施例を説明する。 In this example, examples related to the production and use of the compound of the present disclosure will be described.
（実施例１）５’-アミノ-５’-デオキシヌクレオシドのアミダイト試薬の合成
　薄層クロマトグラフィーはSilicagel　70　TLC　Plate　(富士フィルム和光純薬工業)を用いた。カラムクロマトグラフィーにはＷａｋｏｇｅｌ　Ｃ-２００（富士フィルム和光純薬工業）およびSilica　Gel　60　(ナカライテスク)を用いた。１Ｈ　ＮＭＲおよび１３Ｃ　ＮＭＲはテトラメチルシランを内部標準とし、Bruker　Avance　III　500　MHzを用いて測定した。質量分析は北海道大学グローバルファシリティセンター設置のThermo　Scientific　Exactiveを用いて測定した。 (Example 1) Synthesis of amidite reagent of 5'-amino-5'-deoxynucleoside Silicagel 70 TLC Plate (Fuji Film Wako Pure Chemical Industries, Ltd.) was used for thin layer chromatography. Wakogel C-200 (Fuji Film Wako Pure Chemical Industries) and Silica Gel 60 (Nacalai Tesque) were used for column chromatography. 1 H NMR and 13 C NMR were measured using a Bruker Avance III 500 MHz using tetramethylsilane as an internal standard. Mass spectrometry was measured using Thermo Scientific Exactive installed at Hokkaido University Global Facility Center.
　５’-（４-モノメトキシトリチルアミノ）-２’-フルオロ-２’、５’-ジデオキシウリジン　３’-ホスホロアミダイト（化合物５）の合成
　論文記載（Ueno,　Y.,　Hirai,　M.,　Yoshikawa,　K.,　Kitamura,　Y.,　Hirata,　Y.,　Kiuchi,　K.,　Kitade,　Y.　Tetrahedron,　2008,　64,　11328-11334.）の方法を参考にして、本化合物５を以下に示す合成スキーム、方法により合成した。

スキーム１.Qに2’F-5’-NH2-ウリジンを導入するための試薬（5’-アミノ-2’-フルオロ-2’,5’-ジデオキシウリジン　アミダイト試薬、化合物５）の合成スキーム Synthesis of 5'-(4-monomethoxytritylamino)-2'-fluoro-2',5'-dideoxyuridine 3'-phosphoramidite (compound 5) Paper description (Ueno, Y., Hirai, M., This compound 5 was synthesized as shown below by referring to the method of Yoshikawa, K., Kitamura, Y., Hirata, Y., Kiuchi, K., Kitade, Y. Tetrahedron, 2008, 64, 11328-11334. Synthesized according to the scheme and method.

Scheme 1. Synthesis scheme of reagent for introducing 2'F-5'-NH 2 -uridine into Q (5'-amino-2'-fluoro-2',5'-dideoxyuridine amidite reagent, compound 5)
　３’-O-tert-ブチルジメチルシリル-２’-フルオロ-２’-デオキシウリジン（化合物２）
アルゴン雰囲気下、２’-フルオロ-２’-デオキシウリジン（化合物１）１.２３ｇ（５.００ｍｍｏｌ）をジメチルホルムアミド（５０ｍL）に溶解し、tert-ブチルジメチルクロロシラン２.２６ｇ（１５.０ｍｍｏｌ）およびイミダゾール２.０４ｇ（３０.０ｍｍｏｌ）を加え、室温で２２時間撹拌した。反応液にエタノール（５.０ｍL）を加えて室温で３０分攪拌した後、酢酸エチル（２４０ｍL）を加え、水（７０ｍL）で４回、飽和食塩水（７０ｍL）で１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮して白色泡状物質を得た。本物質を氷冷下、テトラヒドロフラン－トリフルオロ作酸－水（４：１：１）の混合溶液（６０ｍL）に溶解し、氷冷下で２.５時間、さらに室温で２時間攪拌した。反応液に飽和炭酸水素ナトリウム水溶液（１００ｍL）を加えた後、酢酸エチル（２００ｍL）により抽出した。さらに有機層を飽和炭酸水素ナトリウム水溶液（７０ｍL）、水（７０ｍL）、飽和食塩水（７０ｍL）で各１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：エタノール－クロロホルム）により精製して標記化合物（化合物２）１.２４ｇ（収率６９％）を白色固体状物質として得た。
ESI-MS　計算値：383.14090　(C15H25O5N2FSiNa　[M+Na]+),　実測値：383.14060.
1H　NMR　(500　MHz,　DMSO-d6)　δ:　11.40　(br　s,　1　H,　NH),　7.89　(d,　1　H,　ArH,　J　=　8.1　Hz),　5.90　(d,　1　H,　CH,　J　=　17.8　Hz),　5.64　(d,　1　H,　ArH,　J　=　8.1　Hz),　5.23　(br　s,　1　H,　OH),　5.11　(d,　1　H,　CH,　J　=　53.4　Hz),　4.33　(m,　1　H,　CH),　3.87　(m,　1　H,　CH),　3.76　(m,　1　H,　CH2a),　3.54　(m,　1　H,　CH2b),　0.88　(s,　9　H,　(CH3)3),　0.10　(s,　6　H,　(CH3)2).
13C　NMR　(125　MHz,　DMSO-d6)　δ:　163.69　(C),　150.79　(C),　141.03　(CH),　102.15　(CH),　93.01　(CH,　J　=　187.7　Hz),　87.92　(CH,　J　=　34.4　Hz),　83.92　(CH),　69.19　(CH,　J　=　15.5　Hz),　59.56　(CH2),　26.03　(CH3),　18.23　(C),　-4.45　(CH3),　-4.71　(CH3).　 3'-O-tert-butyldimethylsilyl-2'-fluoro-2'-deoxyuridine (compound 2)
Under an argon atmosphere, 1.23 g (5.00 mmol) of 2'-fluoro-2'-deoxyuridine (compound 1) was dissolved in dimethylformamide (50 mL), and 2.26 g (15.0 mmol) of tert-butyldimethylchlorosilane and 2.04 g (30.0 mmol) of imidazole was added and stirred at room temperature for 22 hours. After adding ethanol (5.0 mL) to the reaction solution and stirring at room temperature for 30 minutes, ethyl acetate (240 mL) was added, washed four times with water (70 mL) and once with saturated brine (70 mL), and washed with sodium sulfate. dried. The solution was concentrated under reduced pressure to give a white foam. This substance was dissolved in a mixed solution (60 mL) of tetrahydrofuran-trifluorinated acid-water (4:1:1) under ice-cooling, and stirred for 2.5 hours under ice-cooling and then for 2 hours at room temperature. A saturated aqueous sodium hydrogen carbonate solution (100 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (200 mL). Furthermore, the organic layer was washed once each with a saturated aqueous sodium bicarbonate solution (70 mL), water (70 mL), and saturated brine (70 mL), and dried over sodium sulfate. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: ethanol-chloroform) to obtain 1.24 g (yield 69%) of the title compound (compound 2) as a white solid substance.
ESI-MS calculated value: 383.14090 (C 15 H 25 O 5 N 2 FSiNa　[M+Na] + ), actual value: 383.14060.
1 H NMR (500 MHz, DMSO-d 6 ) δ: 11.40 (br s, 1 H, NH), 7.89 (d, 1 H, ArH, J = 8.1 Hz), 5.90 (d, 1 H, CH, J = 17.8 Hz), 5.64 (d, 1 H, ArH, J = 8.1 Hz), 5.23 (br s, 1 H, OH), 5.11 (d, 1 H, CH, J = 53.4 Hz), 4.33 (m, 1 H, CH), 3.87 (m, 1 H, CH), 3.76 (m, 1 H, CH 2 a), 3.54 (m, 1 H, CH 2 b), 0.88 (s, 9 H, (CH 3 ) 3 ), 0.10 (s, 6 H, (CH 3 ) 2 ).
13 C NMR (125 MHz, DMSO-d 6 ) δ: 163.69 (C), 150.79 (C), 141.03 (CH), 102.15 (CH), 93.01 (CH, J = 187.7 Hz), 87.92 (CH, J = 34.4 Hz), 83.92 (CH), 69.19 (CH, J = 15.5 Hz), 59.56 (CH 2 ), 26.03 (CH 3 ), 18.23 (C), -4.45 (CH 3 ), -4.71 (CH 3 ).
　５’-アジド-３’-O-（tert-ブチルジメチルシリル）-２’-フルオロ-２’、５’-ジデオキシウリジン（化合物３）
アルゴン雰囲気下、３’-O-tert-ブチルジメチルシリル-２’-フルオロ-２’-デオキシウリジン（化合物２）１.２４ｇ（３.４４ｍｍｏｌ）をピリジン（４０ｍL）に溶解し、p-トルエンスルホニルクロリド１.３１ｇ（６.８８ｍｍｏｌ）を加え、室温で３日間撹拌した。反応液に水（５.０ｍL）を加えて室温で１時間攪拌した後、減圧下濃縮した。残渣に酢酸エチル（１６０ｍL）を加えて溶解し、水（６０ｍL）、飽和炭酸水素ナトリウム水溶液（６０ｍL）、水（６０ｍL）、飽和食塩水（６０ｍL）で各１回洗浄し、硫酸ナトリウ
ムにより乾燥した。溶液を減圧下濃縮した後、残渣をトルエンで共沸して黄色泡状物質を得た。アルゴン雰囲気下、本物質をジメチルホルムアミド（３５ｍL）に溶解し、アジ化ナトリウム６１０ｍｇ（９.３９ｍｍｏｌ）および塩化アンモニウム６７０ｍｇ（１２.５ｍｍｏｌ）を加え、８０℃で１時間加熱撹拌した。反応液を室温まで冷却した後、酢酸エチル（２００ｍL）を加え、水（６０ｍL）で４回、飽和食塩水（６０ｍL）で１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：酢酸エチル－ヘキサン）により精製して標記化合物（化合物３）１.０３ｇ（収率７８％）を白色固体状物質として得た。
ESI-MS　計算値：408.14738　(C15H25O4N5FSiNa　[M+Na]+),　実測値：408.14709.
1H　NMR　(500　MHz,　DMSO-d6)　δ:11.46　(br　s,　1　H,　NH),　7.70　(d,　1　H,　ArH,　J　=　8.0　Hz),　5.83　(d,　1　H,　CH,　J　=　22.4　Hz),　5.67　(d,　1　H,　ArH,　J　=　8.0　Hz),　5.29　(dd,　1　H,　CH,　J　=　53.6,　3.8　Hz),　4.50　(m,　1　H,　CH),　3.95　(m,　1　H,　CH),　3.76　(d,　1　H,　CH2a,　J　=　13.8　Hz),　3.45　(dd,　1　H,　CH2b,　J　=　13.8,　4.6　Hz),　0.88　(s,　9　H,　(CH3)3),　0.12　(s,　3　H,　CH3),　0.11　(s,　3　H,　CH3).
13C　NMR　(125　MHz,　DMSO-d6)　δ:　163.68　(C),　150.65　(C),　142.87　(CH),　102.49　(CH),　92.48　(CH,　J　=　186.1　Hz),　90.50　(CH,　J　=　36.4　Hz),　81.02　(CH),　70.29　(CH,　J　=　15.8　Hz),　50.48　(CH2),　26.02　(CH3),　18.20　(C),　-4.49　(CH3),　-4.75　(CH3). 5'-azido-3'-O-(tert-butyldimethylsilyl)-2'-fluoro-2',5'-dideoxyuridine (compound 3)
Under an argon atmosphere, 1.24 g (3.44 mmol) of 3'-O-tert-butyldimethylsilyl-2'-fluoro-2'-deoxyuridine (compound 2) was dissolved in pyridine (40 mL), and p-toluenesulfonyl 1.31 g (6.88 mmol) of chloride was added, and the mixture was stirred at room temperature for 3 days. Water (5.0 mL) was added to the reaction solution, stirred at room temperature for 1 hour, and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate (160 mL), washed once each with water (60 mL), saturated aqueous sodium bicarbonate solution (60 mL), water (60 mL), and saturated brine (60 mL), and dried over sodium sulfate. . After the solution was concentrated under reduced pressure, the residue was azeotroped with toluene to give a yellow foam. This substance was dissolved in dimethylformamide (35 mL) under an argon atmosphere, 610 mg (9.39 mmol) of sodium azide and 670 mg (12.5 mmol) of ammonium chloride were added, and the mixture was heated and stirred at 80° C. for 1 hour. After the reaction solution was cooled to room temperature, ethyl acetate (200 mL) was added, washed four times with water (60 mL) and once with saturated brine (60 mL), and dried over sodium sulfate. The solution was concentrated under reduced pressure and purified by silica gel column chromatography (elution solvent: ethyl acetate-hexane) to obtain 1.03 g (yield 78%) of the title compound (compound 3) as a white solid substance.
ESI-MS calculated value: 408.14738 (C 15 H 25 O 4 N 5 FSiNa　[M+Na] + ), actual value: 408.14709.
1 H NMR (500 MHz, DMSO-d 6 ) δ:11.46 (br s, 1 H, NH), 7.70 (d, 1 H, ArH, J = 8.0 Hz), 5.83 (d, 1 H, CH, J = 22.4 Hz), 5.67 (d, 1 H, ArH, J = 8.0 Hz), 5.29 (dd, 1 H, CH, J = 53.6, 3.8 Hz), 4.50 (m, 1 H, CH), 3.95 (m , 1 H, CH), 3.76 (d, 1 H, CH 2 a, J = 13.8 Hz), 3.45 (dd, 1 H, CH 2 b, J = 13.8, 4.6 Hz), 0.88 (s, 9 H, (CH 3 ) 3 ), 0.12 (s, 3 H, CH 3 ), 0.11 (s, 3 H, CH 3 ).
13 C NMR (125 MHz, DMSO-d 6 ) δ: 163.68 (C), 150.65 (C), 142.87 (CH), 102.49 (CH), 92.48 (CH, J = 186.1 Hz), 90.50 (CH, J = 36.4 Hz), 81.02 (CH), 70.29 (CH, J = 15.8 Hz), 50.48 (CH 2 ), 26.02 (CH 3 ), 18.20 (C), -4.49 (CH 3 ), -4.75 (CH 3 ).
　５’-（４-モノメトキシトリチルアミノ）-２’-フルオロ-２’、５’-ジデオキシウリジン（化合物４）
５’-アジド-３’-O-（tert-ブチルジメチルシリル）-２’-フルオロ-２’、５’-ジデオキシウリジン（化合物３）１.０３ｇ（２.６７ｍｍｏｌ）をテトラヒドロフラン－水（９：１）の混合溶液（４０ｍL）に溶解し、トリメチルホスフィン（３mol/L　テトラヒドロフラン溶液）１.７８ｍL（５.３４ｍｍｏｌ）を加え、室温で１６時間撹拌した。反応液に１N水酸化ナトリウム溶液（２.７ｍL）を加えて室温で１時間攪拌した後、減圧下濃縮した。反応液に水（７５ｍL）および飽和炭酸水素ナトリウム水溶液（７５ｍL）を加えた後、酢酸エチル（６０ｍL）で３回抽出した。さらに有機層を飽和食塩水（６０ｍL）で１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮して白色固体状物質を得た。アルゴン雰囲気下、本物質をピリジン（３５ｍL）に溶解し、４-モノメトキシトリチルクロリド１.６５ｇ（５.３４ｍｍｏｌ）を加え、室温で１８時間撹拌した。反応液にエタノール（３.０ｍL）を加えて室温で３０分攪拌した後、減圧下濃縮した。残渣に酢酸エチル（１５０ｍL）を加えて溶解し、水（５０ｍL）で１回、飽和炭酸水素ナトリウム水溶液（５０ｍL）で２回、飽和食塩水（６０ｍL）で１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮した後、残渣をトルエンで共沸して黄色泡状物質を得た。アルゴン雰囲気下、本物質をテトラヒドロフラン（３５ｍL）に溶解し、氷冷下でテトラブチルアンモニウムフルオリド（１mol/L　テトラヒドロフラン溶液）３.７４ｍL（３.７４ｍｍｏｌ）を加えて、氷冷下１時間撹拌した。反応液に酢酸（０.２１ｍL）を加えた後、減圧下濃縮し、残渣をシリカゲルカラムクロマトグラフィー（溶出溶媒：エタノール－クロロホルム、０.１％ピリジンを含む）により精製して標記化合物（化合物４）９９６ｍｇ（収率
７２％）を白色泡状物質として得た。
ESI-MS　計算値：540.19052　(C29H28O5N3FNa　[M+Na]+),　実測値：540.19032.1H　NMR　(500　MHz,　DMSO-d6)　δ:　11.43　(br　s,　1　H,　NH),　7.71　(d,　1　H,　ArH,　J　=　8.1　Hz),　7.41　(d,　4　H,　ArH,　J　=　7.7　Hz),　7.30-7.27　(m,　6　H,　ArH),　7.18　(t,　2　H,　ArH,　J　=　7.3　Hz),　6.85　(d,　2　H,　ArH,　J　=　8.7　Hz),　5.85　(d,　1　H,　CH,　J　=　20.9　Hz),　5.57　(d,　1　H,　ArH,　J　=　8.1　Hz),　5.51　(d,　1　H,　OH,　J　=　6.7　Hz),　5.11　(dd,　1　H,　CH,　J　=　53.5,　3.9　Hz),　4.18　(m,　1　H,　CH),　3.95　(m,　1　H,　CH),　3.72　(s,　3　H,　OCH3),　2.70　(t,　1　H,　NH,　J　=　8.0　Hz),　2.40　(m,　1　H,　CH2a),　2.27　(m,　1　H,　CH2b).
13C　NMR　(125　MHz,　DMSO-d6)　δ:　163.65　(C),　157.89　(C),　150.61　(C),　146.63　(C),　142.12　(CH),　138.16　(C),　130.09　(CH),　129.37　(CH),　128.77　(CH),　128.68　(CH),　128.22　(CH),　126.56　(CH),　113.56　(CH),　102.14　(CH),　93.81　(CH,　J　=　183.8　Hz),　89.57　(CH,　J　=　35.6　Hz),　82.03　(CH),　70.18　(C),　69.98　(CH,　J　=　16.2　Hz),　55.44　(CH3),　45.37　(CH2). 5'-(4-monomethoxytritylamino)-2'-fluoro-2',5'-dideoxyuridine (compound 4)
1.03 g (2.67 mmol) of 5'-azido-3'-O-(tert-butyldimethylsilyl)-2'-fluoro-2',5'-dideoxyuridine (compound 3) was added to tetrahydrofuran-water (9: 1) was dissolved in the mixed solution (40 mL), 1.78 mL (5.34 mmol) of trimethylphosphine (3 mol/L tetrahydrofuran solution) was added, and the mixture was stirred at room temperature for 16 hours. A 1N sodium hydroxide solution (2.7 mL) was added to the reaction solution, stirred at room temperature for 1 hour, and then concentrated under reduced pressure. After adding water (75 mL) and a saturated aqueous sodium hydrogen carbonate solution (75 mL) to the reaction solution, the mixture was extracted three times with ethyl acetate (60 mL). Furthermore, the organic layer was washed once with saturated brine (60 mL) and dried over sodium sulfate. The solution was concentrated under reduced pressure to obtain a white solid material. This substance was dissolved in pyridine (35 mL) under an argon atmosphere, 1.65 g (5.34 mmol) of 4-monomethoxytrityl chloride was added, and the mixture was stirred at room temperature for 18 hours. Ethanol (3.0 mL) was added to the reaction solution, stirred at room temperature for 30 minutes, and then concentrated under reduced pressure. The residue was dissolved in ethyl acetate (150 mL), washed once with water (50 mL), twice with saturated aqueous sodium bicarbonate solution (50 mL), once with saturated brine (60 mL), and dried over sodium sulfate. . After the solution was concentrated under reduced pressure, the residue was azeotroped with toluene to give a yellow foam. This substance was dissolved in tetrahydrofuran (35 mL) under an argon atmosphere, and 3.74 mL (3.74 mmol) of tetrabutylammonium fluoride (1 mol/L tetrahydrofuran solution) was added under ice cooling, and the mixture was stirred for 1 hour under ice cooling. . After adding acetic acid (0.21 mL) to the reaction solution, it was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (elution solvent: ethanol-chloroform, containing 0.1% pyridine) to obtain the title compound (Compound 4). ) 996 mg (yield 72%) were obtained as a white foam.
ESI-MS calculated value: 540.19052 (C 29 H 28 O 5 N 3 FNa　[M+Na] + ), Actual value: 540.19032. 1 H NMR (500 MHz, DMSO-d 6 ) δ: 11.43 (br s, 1 H, NH), 7.71 (d, 1 H, ArH, J = 8.1 Hz), 7.41 (d, 4 H, ArH, J = 7.7 Hz), 7.30-7.27 (m, 6 H, ArH), 7.18 (t, 2 H, ArH, J = 7.3 Hz), 6.85 (d, 2 H, ArH, J = 8.7 Hz), 5.85 (d, 1 H, CH, J = 20.9 Hz), 5.57 (d, 1 H, ArH, J = 8.1 Hz), 5.51 (d, 1 H, OH, J = 6.7 Hz), 5.11 (dd, 1 H, CH, J = 53.5, 3.9 Hz), 4.18 (m, 1 H, CH), 3.95 (m, 1 H, CH), 3.72 (s, 3 H, OCH 3 ), 2.70 (t, 1 H, NH, J = 8.0 Hz), 2.40 (m, 1 H, CH 2 a), 2.27 (m, 1 H, CH 2 b).
13 C NMR (125 MHz, DMSO-d 6 ) δ: 163.65 (C), 157.89 (C), 150.61 (C), 146.63 (C), 142.12 (CH), 138.16 (C), 130.09 (CH), 129.37 (CH), 128.77 (CH), 128.68 (CH), 128.22 (CH), 126.56 (CH), 113.56 (CH), 102.14 (CH), 93.81 (CH, J = 183.8 Hz), 89.57 (CH, J = 35.6 Hz), 82.03 (CH), 70.18 (C), 69.98 (CH, J = 16.2 Hz), 55.44 (CH 3 ), 45.37 (CH 2 ).
　５’-（４-モノメトキシトリチルアミノ）-２’-フルオロ-２’、５’-ジデオキシウリジン　３’-ホスホロアミダイト（化合物５）アルゴン雰囲気下、５’-（４-モノメトキシトリチルアミノ）-２’-フルオロ-２’、５’-ジデオキシウリジン（化合物４）５００ｍｇ（０.９７ｍｍｏｌ）を塩化メチレン（２０ｍL）に溶解し、2-シアノエチルジイソプロピルクロロホスホロアミジト０.５４ｍL（１.９４ｍｍｏｌ）およびジイソプロピルエチルアミン０.６６ｍL（３.８８ｍｍｏｌ）を加え、室温で５０分撹拌した。反応液にクロロホルム（８０ｍL）を加え、飽和炭酸水素ナトリウム水溶液（３０ｍL）、水（３０ｍL）、飽和食塩水（３０ｍL）で各１回洗浄し、硫酸ナトリウムにより乾燥した。溶液を減圧下濃縮後、シリカゲルカラムクロマトグラフィー（溶出溶媒：酢酸エチル－ヘキサン、０.１％ピリジンを含む）により精製して標記化合物（化合物５）５２０ｍｇ（収率７５％）を白色泡状物質として得た。
ESI-MS　計算値：740.29837　(C38H45O6N5FPNa　[M+Na]+),　実測値：740.29836.
31P　NMR　(202　MHz,　DMSO-d6)　δ:　149.45　(d,　J　=　6.9　Hz),　149.23　(d,　J　=　11.9　Hz). 5'-(4-monomethoxytritylamino)-2'-fluoro-2', 5'-dideoxyuridine 3'-phosphoramidite (compound 5) under argon atmosphere, 5'-(4-monomethoxytritylamino) -2'-Fluoro-2',5'-dideoxyuridine (Compound 4) 500 mg (0.97 mmol) was dissolved in methylene chloride (20 mL), and 2-cyanoethyldiisopropylchlorophosphoroamidite 0.54 mL (1.94 mmol) was dissolved in methylene chloride (20 mL). ) and 0.66 mL (3.88 mmol) of diisopropylethylamine were added, and the mixture was stirred at room temperature for 50 minutes. Chloroform (80 mL) was added to the reaction solution, washed once each with saturated aqueous sodium hydrogen carbonate solution (30 mL), water (30 mL), and saturated brine (30 mL), and dried over sodium sulfate. After concentrating the solution under reduced pressure, it was purified by silica gel column chromatography (elution solvent: ethyl acetate-hexane, containing 0.1% pyridine) to obtain 520 mg (yield 75%) of the title compound (Compound 5) as a white foamy substance. obtained as.
ESI-MS calculated value: 740.29837 (C 38 H 45 O 6 N 5 FPNa　[M+Na] + ), actual value: 740.29836.
31P NMR (202 MHz, DMSO- d6 ) δ: 149.45 (d, J = 6.9 Hz), 149.23 (d, J = 11.9 Hz).
　２’-O-（tert-ブチルジメチルシリル）-５’-（４-モノメトキシトリチルアミノ）-５’-デオキシウリジン　３’-ホスホロアミダイト（化合物６）の合成

スキーム２.　Qに2’OH-5’-NH2-ウリジンを導入するための試薬（5’-アミノ-5’-デオキシヌクレオシド　アミダイト試薬、化合物６）の構造
　本化合物６は、論文記載（Kojima,　N.　and　Bruice,　T.　C.　Org.Lett.,　2000,　2,　81-84.、Kojima,　N.,　Szabo,　I.　E.,　Bruice,　T.　C.　Tetrahedron,　2002,　58,　867-879.、Maruyama,　H.,　Oikawa,　R.,　Hayakawa,　M.,　Takamori,　S.,　Kimura,　Y.,　Abe,　N.,　Tsuji,　G.,　Matsuda,　A.,　Shuto,　S.,　Ito,　Y.,　Abe,　H.　Nucleic　Acids　Res.,　2017,　45,　7042-7048.）の方法に従い合成した。 Synthesis of 2'-O-(tert-butyldimethylsilyl)-5'-(4-monomethoxytritylamino)-5'-deoxyuridine 3'-phosphoramidite (compound 6)

Scheme 2. Structure of a reagent for introducing 2'OH-5'-NH 2 -uridine into Q (5'-amino-5'-deoxynucleoside amidite reagent, compound 6) Compound 6 is the structure described in the paper (Kojima , N. and Bruice, T. C. Org.Lett., 2000, 2, 81-84., Kojima, N., Szabo, I. E., Bruice, T. C. Tetrahedron, 2002, 58, 867-879., Maruyama, H., Oikawa , R., Hayakawa, M., Takamori, S., Kimura, Y., Abe, N., Tsuji, G., Matsuda, A., Shuto, S., Ito, Y., Abe, H. Nucleic Acids Res., 2017, 45, 7042-7048.).
　合成したオリゴヌクレオチド（ＤＮＡ、及びＲＮＡ）は、高速液体クロマトグラフィー（ＨＰＬＣ）によって精製した。ＨＰＬＣは、Ｇｉｌｓｏｎ社の装置にＷａｔｅｒｓ　μ－Ｂｏｎｄａｓｐｈｅｒｅ　Ｃ１８　３００Ａ（内径３．９ｍｍ×長さ１５０ｍｍ、Ｗａｔｅｒｓ社）を接続して行った。移動相として、逆相の場合には０．１Ｍ　酢酸トリエチルアンモニウム緩衝液（ＴＥＡＡ、ｐＨ７．０）中アセトニトリルの濃度勾配を用いた。合成したオリゴヌクレオチドの種類と逆相ＨＰＬＣの条件を下記に示す。
　Ａ溶液：５％アセトニトリル／０．１Ｍ　ＴＥＡＡ（ｐＨ７．０）
　Ｂ溶液：２５％アセトニトリル／０．１Ｍ　ＴＥＡＡ（ｐＨ７．０） The synthesized oligonucleotides (DNA and RNA) were purified by high performance liquid chromatography (HPLC). HPLC was performed by connecting a Waters μ-Bondasphere C18 300A (inner diameter 3.9 mm x length 150 mm, Waters) to a Gilson device. As mobile phase, a concentration gradient of acetonitrile in 0.1 M triethylammonium acetate buffer (TEAA, pH 7.0) was used in the case of reversed phase. The types of oligonucleotides synthesized and the conditions for reverse phase HPLC are shown below.
Solution A: 5% acetonitrile/0.1M TEAA (pH 7.0)
Solution B: 25% acetonitrile/0.1M TEAA (pH 7.0)
５’-（４-モノメトキシトリチルアミノ）-２’-フルオロ-２’、５’-ジデオキシシチジン　３’-ホスホロアミダイト（化合物１０）の合成
論文記載（Hideto　Maruyama　et　al.,Nucleic　Acids　Research,　2017,　Vol.　45,　No.　12，7042-7048）の方法を参考にして、本化合物１０を以下に示す合成スキーム、方法により合成した。

スキーム３.　Qに2’F-5’-NH2-シチジンを導入するための試薬（5’-アミノ-2’-フルオロ-2’,5’-ジデオキシシチジン　アミダイト試薬、化合物１０）の合成スキーム Synthesis of 5'-(4-monomethoxytritylamino)-2'-fluoro-2',5'-dideoxycytidine 3'-phosphoramidite (Compound 10) (Hideto Maruyama et al., Nucleic Acids Research, 2017, Vol. 45, No. 12, 7042-7048), the present compound 10 was synthesized using the synthesis scheme and method shown below.

Scheme 3. Synthesis scheme of reagent for introducing 2'F-5'-NH 2 -cytidine into Q (5'-amino-2'-fluoro-2',5'-dideoxycytidine amidite reagent, compound 10)
２’-O-（tert-ブチルジメチルシリル）-５’-（４-モノメトキシトリチルアミノ）-５’-デオキシシチジン　３’-ホスホロアミダイト（化合物１１）の合成

スキーム４.　Qに2’OH-5’-NH2-シチジンを導入するための試薬（5’-アミノ-5’-デオキシシチジン　アミダイト試薬、化合物１１）の構造
　本化合物１１は、論文記載（Afaf　H.　El-Sagheer　et　al.,　Chem.　Commun.,　2017,　53,　10700-10702、　Afaf　H.　El-Sagheer　et　al.,　PNAS　2010　107　15329-15334、Afaf　H.　El-Sagheer　et　al.,　PNAS,　2011,　108,　11338-11343）の方法を参考にして合成した。 Synthesis of 2'-O-(tert-butyldimethylsilyl)-5'-(4-monomethoxytritylamino)-5'-deoxycytidine 3'-phosphoramidite (Compound 11)

Scheme 4. Structure of a reagent for introducing 2'OH-5'-NH 2 -cytidine into Q (5'-amino-5'-deoxycytidine amidite reagent, compound 11) Compound 11 is the structure described in the paper (Afaf H. El-Sagheer et al., Chem. Commun., 2017, 53, 10700-10702, Afaf H. El-Sagheer et al., PNAS 2010 107 15329-15334, Afaf H. El-Sagheer et al., PNAS , 2011, 108, 11338-11343).
（ＤＮＦＢ修飾体（２Ｆｐｓ―ＤＮＦＢ）（実施例２）の作製）
２ＦｐｓのDNFB修飾体（実施例２）は以下の方法で作製した。３’－ホスホロチオエート修飾を含む核酸鎖２Ｆｐｓ（図３）（１．０ｎｍｏｌ）を１０ｍM　１－フルオロ－２,４－ジニトロベンゼン（ＤＮＦＢ）、１０％　ｄｉｍｅｔｈｙｌ　ｓｕｌｆｏｘｉｄｅ（ＤＭＳＯ）、及び２０ｍＭ　ホウ酸ナトリウム(ｐH８．５)に溶解した。室温で２時間反応を行った後、ゲルろ過カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ、ＮＡＰ－５）を行って粗精製した。その後、逆相カラムを用いたＨＰＬＣを用いて精製し、２ＦｐｓのＤＮＦＢ修飾体（２Ｆｐｓ―ＤＮＦＢ）を得た。精製には下記の溶液を用いた。
　Ａ溶液：５％アセトニトリル／０．１Ｍ　ＴＥＡＡ（ｐＨ７．０）
　Ｂ溶液：５０％アセトニトリル／０．１Ｍ　ＴＥＡＡ（ｐＨ７．０）
より具体的には図６に示すように、実施例２の２ＦｐｓのＤＮＦＢ修飾体は、原料である２Ｆｐｓよりも遅れてピークが観察されることが確認された。 (Preparation of modified DNFB (2Fps-DNFB) (Example 2))
A 2 Fps DNFB modified product (Example 2) was produced by the following method. Nucleic acid strand 2Fps (Figure 3) (1.0 nmol) containing 3'-phosphorothioate modification was mixed with 10 mM 1-fluoro-2,4-dinitrobenzene (DNFB), 10% dimethyl sulfoxide (DMSO), and 20 mM sodium borate (pH 8). .5). After reaction at room temperature for 2 hours, crude purification was performed using a gel filtration column (GE Healthcare, NAP-5). Thereafter, it was purified using HPLC using a reverse phase column to obtain a 2Fps modified DNFB (2Fps-DNFB). The following solution was used for purification.
Solution A: 5% acetonitrile/0.1M TEAA (pH 7.0)
Solution B: 50% acetonitrile/0.1M TEAA (pH 7.0)
More specifically, as shown in FIG. 6, it was confirmed that the 2Fps modified DNFB of Example 2 had a peak observed later than that of the raw material, 2Fps.
２ＭｅｐｓのＤＮＦＢ修飾体は３’－ホスホロチオエート修飾を含む核酸鎖２Ｍｅｐｓ（図３）を用い、上記２Ｆｐｓ―ＤＮＦＢ（実施例２）と同様の方法で反応を行い、作製した。 The DNFB-modified product of 2Meps was prepared by using the nucleic acid chain 2Meps (FIG. 3) containing 3'-phosphorothioate modification and performing a reaction in the same manner as the above-mentioned 2Fps-DNFB (Example 2).
（ＲＮＡの連結体（２Ｍｅ―２ＯＨ５Ｎ）（実施例３）の作製）
　連結体の２Ｍｅ―２ＯＨ５Ｎ（１０－１３）は以下の方法で作製した。５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（２．５ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７（図３）（５ｐｍｏｌ）とを２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。ＤＮＦＢ修飾を含む核酸鎖２Ｍｅｐｓ―ＤＮＦＢ（２５ｐｍｏｌ）を２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解し、反応液１に加え、総量２５μＬで２７℃、１０、３０、６０、及び１２０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）５μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、蛍光イメージングを用いて分析した。より具体的には、図６に示すように、実施例３の連結体は、原料である蛍光分子で修飾された核酸鎖２ＯＨ５Ｎの核酸鎖よりも遅れて移動することが確認された。また反応時間の経過に伴い、観察される連結体の量は増加した。ゲルの分析は電気泳動後にＴｙｐｈｏｏｎ　ＦＬＡ９０００（ＧＥ　Ｈｅａｌｔｈ．）によって解析した。 (Preparation of RNA conjugate (2Me-2OH5N) (Example 3))
The conjugate 2Me-2OH5N (10-13) was produced by the following method. Nucleic acid chain 2OH5N (Figure 3) (2.5 pmol) containing 5' amino modification and modified with a fluorescent molecule and template nucleic acid chain tmr27 (Figure 3) (5 pmol) were mixed in 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8). ) (total volume: 12.5 μL). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Nucleic acid chain 2Meps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 μL) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 μL. After reacting for 30, 60, and 120 minutes, 5 μL was mixed with 5 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging. More specifically, as shown in FIG. 6, it was confirmed that the conjugate of Example 3 migrated later than the 2OH5N nucleic acid chain modified with a fluorescent molecule as a raw material. Furthermore, the amount of conjugates observed increased as the reaction time progressed. Gel analysis was performed using Typhoon FLA9000 (GE Health.) after electrophoresis.
（ＲＮＡの連結体（２Ｍｅ―２Ｆ５Ｎ））（実施例４）の作製）
　連結体の２Ｍｅ―２Ｆ５Ｎ（１０－１３）（実施例４、図３）は以下の方法で作製した。５’アミノ修飾を含み含蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（２．５ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７（図３）（５ｐｍｏｌ）とを２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。ＤＮＦＢ修飾を含む核酸鎖２Ｍｅｐｓ―ＤＮＦＢ（２５ｐｍｏｌ）を２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解し、反応液１に加え、総量２５μＬで２７℃、１０、３０、６０、及び１２０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）５μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、上記２Ｍｅ―２ＯＨ５Ｎ（実施例４）と同様の方法で蛍光イメージングを用いて解析した。より具体的には、図６Ａに示すように、実施例４の連結体は、原料である蛍光分子で修飾された核酸鎖２Ｆ５Ｎの核酸鎖よりも遅れて移動することが確認された。また図６Ｂに示すように、反応時間の経過に伴い、観察される連結体の反応収量は増加し、いずれの時間においても上記２Ｍｅ―２ＯＨ５Ｎよりも高い反応収量を示したことから、２Ｆ５ＮはＤＮＦＢ修飾核酸との連結反応において２ＯＨ５Ｎよりも有効であることを確認した。 (Preparation of RNA conjugate (2Me-2F5N)) (Example 4))
The conjugate 2Me-2F5N(10-13) (Example 4, FIG. 3) was produced by the following method. A nucleic acid chain 2OH5N (Figure 3) (2.5 pmol) containing a 5' amino modification and modified with a fluorescent molecule and a template nucleic acid chain tmr27 (Figure 3) (5 pmol) were mixed in 20 mM sodium phosphate, 10 mM magnesium chloride ( pH 8) (total volume: 12.5 μL). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Nucleic acid chain 2Meps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 μL) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 μL. After reacting for 30, 60, and 120 minutes, 5 μL was mixed with 5 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 4). . More specifically, as shown in FIG. 6A, it was confirmed that the conjugate of Example 4 migrated later than the nucleic acid strand of the fluorescent molecule-modified nucleic acid strand 2F5N, which was the raw material. Furthermore, as shown in Figure 6B, the observed reaction yield of the conjugate increased as the reaction time progressed, and the reaction yield was higher than that of the 2Me-2OH5N at any time, so 2F5N was It was confirmed that it is more effective than 2OH5N in ligation reactions with modified nucleic acids.
（ＲＮＡの連結体（２Ｆ―２ＯＨ５Ｎ）（実施例５）の作製）
　　連結体の２Ｆ―２ＯＨ５Ｎは以下の方法で作製した。５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（２．５ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７（図３）（５ｐｍｏｌ）とを２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。ＤＮＦＢ修飾を含む核酸鎖２Ｆｐｓ―ＤＮＦＢ（２５ｐｍｏｌ）を２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解し、反応液１に加え、総量２５μＬで２７℃、１０、３０、６０、及び１２０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）５μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、上記２Ｍｅ―２ＯＨ５Ｎ（実施例５）と同様の方法で蛍光イメージングを用いて解析した。より具体的には、図６Ａに示すように、実施例５の連結体は、原料である蛍光分子で修飾された核酸鎖２ＯＨ５Ｎの核酸鎖よりも遅れて移動することが確認された。 (Preparation of RNA conjugate (2F-2OH5N) (Example 5))
The conjugate 2F-2OH5N was produced by the following method. A nucleic acid chain 2OH5N (Figure 3) (2.5 pmol) containing a 5' amino modification and modified with a fluorescent molecule and a template nucleic acid chain tmr27 (Figure 3) (5 pmol) were mixed in 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8). ) (total volume: 12.5 μL). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Nucleic acid chain 2Fps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 μL) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 μL. After reacting for 30, 60, and 120 minutes, 5 μL was mixed with 5 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6 M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 5). . More specifically, as shown in FIG. 6A, it was confirmed that the conjugate of Example 5 migrated later than the 2OH5N nucleic acid strand modified with a fluorescent molecule as a raw material.
　（ＲＮＡの連結体（２Ｆ―２Ｆ５Ｎ））（実施例６）の作製）
　連結体の２Ｆ―２Ｆ５Ｎ（実施例６、図３）は以下の方法で作製した。５’アミノ修飾を含み含蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（２．５ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７（図３）（５ｐｍｏｌ）とを２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。ＤＮＦＢ修飾を含む核酸鎖２Ｆｐｓ―ＤＮＦＢ（２５ｐｍｏｌ）を２０ｍＭリン酸ナトリウム、１０ｍＭ塩化マグネシウム（ｐＨ８）を含む溶液（総量１２．５μＬ）に溶解し、反応液１に加え、総量２５μＬで２７℃、１０、３０、６０、及び１２０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）５μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。より具体的には、図６Ａに示すように、実施例６の連結体は、原料である蛍光分子で修飾された核酸鎖２Ｆ５Ｎの核酸鎖よりも遅れて移動することが確認された。また図６Ｂに示すように、反応時間の経過に伴い、観察される連結体の量は増加し、いずれの時間においても上記２Ｆ―２ＯＨ５Ｎよりも高い反応収量を示したことから、２Ｆ５ＮはＤＮＦＢ修飾核酸との連結反応において２ＯＨ５Ｎよりも有効であることを確認した。 (Preparation of RNA concatenation (2F-2F5N) (Example 6))
The conjugate 2F-2F5N (Example 6, FIG. 3) was produced by the following method. A nucleic acid chain 2OH5N (Figure 3) (2.5 pmol) containing a 5' amino modification and modified with a fluorescent molecule and a template nucleic acid chain tmr27 (Figure 3) (5 pmol) were mixed in 20 mM sodium phosphate, 10 mM magnesium chloride ( pH 8) (total volume: 12.5 μL). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Nucleic acid chain 2Fps-DNFB (25 pmol) containing DNFB modification was dissolved in a solution (total volume 12.5 μL) containing 20 mM sodium phosphate and 10 mM magnesium chloride (pH 8), added to reaction solution 1, and incubated at 27°C for 10 minutes in a total volume of 25 μL. After reacting for 30, 60, and 120 minutes, 5 μL was mixed with 5 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). . More specifically, as shown in FIG. 6A, it was confirmed that the conjugate of Example 6 migrated later than the 2F5N nucleic acid strand modified with a fluorescent molecule as a raw material. Furthermore, as shown in Figure 6B, the amount of conjugates observed increased with the passage of reaction time, and the reaction yield was higher than that of the above 2F-2OH5N at all times, so 2F5N was modified with DNFB. It was confirmed that it is more effective than 2OH5N in ligation reactions with nucleic acids.
（ＤＮＡテンプレートを利用したＲＮＡの連結体（実施例７）の作製）
上記２Ｍｅ―２ＯＨ５Ｎ、２Ｍ―２Ｆ５Ｎ、２Ｆ―２ＯＨ５Ｎ、２Ｆ―２Ｆ５ＮをＲＮＡテンプレートｔｍｒ２７の代わりに、ＤＮＡテンプレートｔｍｄ２７を添加して同様に作製した。図８に示すように、ＲＮＡテンプレートと同様に２Ｍｅ―２ＯＨ５Ｎと比較して２Ｍ―２Ｆ５Ｎは高い反応収量を示し、また、２Ｆ―２ＯＨ５Ｎと比較して２Ｆ―２Ｆ５Ｎは高い反応収量を示したことから、２Ｆ５ＮはＤＮＦＢ修飾核酸との連結反応において２ＯＨ５Ｎよりも有効であることを確認した。 (Preparation of RNA conjugate (Example 7) using DNA template)
The above-mentioned 2Me-2OH5N, 2M-2F5N, 2F-2OH5N, and 2F-2F5N were similarly produced by adding DNA template tmd27 instead of RNA template tmr27. As shown in Figure 8, similar to the RNA template, 2M-2F5N showed a higher reaction yield compared to 2Me-2OH5N, and 2F-2F5N showed a higher reaction yield compared to 2F-2OH5N. It was confirmed that 2F5N is more effective than 2OH5N in the ligation reaction with DNFB-modified nucleic acids.
　（活性化剤(ＤＮＦＢ)を利用したＲＮＡの連結体（２Ｍｅ―２ＯＨ５Ｎ）（実施例８）の作製）
　ＤＮＦＢを利用して連結体の２Ｍｅ―２ＯＨ５Ｎ（実施例８、図３）を以下の方法で作製した。３’－ホスホロチオエート修飾を含む核酸鎖２Ｍｅｐｓ（図３）（５０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（１０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７（２０ｐｍｏｌ）とを５０ｍＭリン酸ナトリウム、１００ｍＭ塩化ナトリウム（ｐＨ８）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。１００ｍＭ　ＤＮＦＢを含むＤＭＳＯ溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６、及び２４時間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。より具体的には、図9Ａに示すように、反応時間の経過に伴い観察される連結体の反応量は増加した。 (Preparation of RNA conjugate (2Me-2OH5N) (Example 8) using activator (DNFB))
Using DNFB, a conjugate of 2Me-2OH5N (Example 8, FIG. 3) was produced by the following method. Nucleic acid strand 2Meps containing 3'-phosphorothioate modification (Figure 3) (50 pmol), nucleic acid strand 2OH5N containing 5' amino modification and modified with a fluorescent molecule (Figure 3) (10 pmol), and template nucleic acid strand tmr27 (20 pmol) was dissolved in a solution (total volume 18 μL) containing 50 mM sodium phosphate and 100 mM sodium chloride (pH 8). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Add 2 μL of DMSO solution containing 100 mM DNFB to reaction solution 1, react in a total volume of 20 μL at 27°C for 6 and 24 hours, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to prepare the conjugate. A solution containing was obtained. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). . More specifically, as shown in FIG. 9A, the amount of conjugate reacted increased as the reaction time progressed.
　（活性化剤(ＤＮＦＢ)を利用したＲＮＡの連結体（２Ｍｅ―２Ｆ５Ｎ）（実施例９）の作製）
　　　ＤＮＦＢを利用して連結体の２Ｍｅ―２Ｆ５Ｎ（実施例９、図３）を以下の方法で作製した。３’－ホスホロチオエート修飾を含む核酸鎖２Ｍｅｐｓ（図３）（５０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２Ｆ５Ｎ（図３）（１０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７（２０ｐｍｏｌ）とを５０ｍＭリン酸ナトリウム、１００ｍＭ塩化ナトリウム（ｐＨ８）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。１００ｍＭ　ＤＮＦＢを含むＤＭＳＯ溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６、及び２４時間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。より具体的には、図9Ａに示すように、反応時間の経過に伴い観察される連結体の反応量は増加した。 (Preparation of RNA conjugate (2Me-2F5N) (Example 9) using activator (DNFB))
A conjugate of 2Me-2F5N (Example 9, FIG. 3) was produced by the following method using DNFB. Nucleic acid strand 2Meps containing 3'-phosphorothioate modification (Fig. 3) (50 pmol), nucleic acid strand 2F5N containing 5' amino modification and modified with a fluorescent molecule (Fig. 3) (10 pmol), and template nucleic acid strand tmr27 (20 pmol) was dissolved in a solution (total volume 18 μL) containing 50 mM sodium phosphate and 100 mM sodium chloride (pH 8). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Add 2 μL of DMSO solution containing 100 mM DNFB to reaction solution 1, react in a total volume of 20 μL at 27°C for 6 and 24 hours, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to prepare the conjugate. A solution containing was obtained. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). . More specifically, as shown in FIG. 9A, the amount of conjugate reacted increased as the reaction time progressed.
（ＤＮＡテンプレート及び活性化剤(ＤＮＦＢ)を利用したＲＮＡの連結体（実施例１０）の作製）
ＤＮＦＢを活性化剤として利用して、上記２Ｍｅ―２ＯＨ５Ｎ、および２Ｍｅ―２Ｆ５Ｎを、ＤＮＡテンプレートｔｍｄ２７を用いて同様に作製した。図9Ｂに示すように、ＲＮＡテンプレートと同様に２Ｍｅ―２ＯＨ５Ｎと比較して２Ｍｅ―２Ｆ５Ｎは高い反応収量を示し、また、２Ｆ―２ＯＨ５Ｎと比較して２Ｆ―２Ｆ５Ｎは高い反応収量を示したことから、２Ｆ５ＮはＤＮＦＢを用いた連結反応において２ＯＨ５Ｎよりも有効であることを確認した。 (Preparation of RNA conjugate (Example 10) using DNA template and activator (DNFB))
Using DNFB as an activator, the above 2Me-2OH5N and 2Me-2F5N were similarly produced using DNA template tmd27. As shown in Figure 9B, similar to the RNA template, 2Me-2F5N showed a higher reaction yield compared to 2Me-2OH5N, and 2F-2F5N showed a higher reaction yield compared to 2F-2OH5N. , 2F5N was confirmed to be more effective than 2OH5N in ligation reactions using DNFB.
　（活性化剤（ＤＮＦＢ）を利用したＲＮＡの連結体（２Ｍｅ―２ＯＨ５Ｎ＿Ｃ）（実施例１１）の作製）
　ＤＮＦＢを利用して連結体の２Ｍｅ―２ＯＨ５Ｎ＿Ｃ（実施例１１、図３）を以下の方法で作製した。３’－ホスホロチオエート修飾を含む核酸鎖２Ｍｅｐｓ（図３）（５０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ＿Ｃ（図３）（１０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７－ＩＩ（図３）（２０ｐｍｏｌ）とを５０ｍＭリン酸ナトリウム、１００ｍＭ塩化ナトリウム（ｐＨ８）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。１００ｍＭ　ＤＮＦＢを含むＤＭＳＯ溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６時間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。 (Preparation of RNA conjugate (2Me-2OH5N_C) (Example 11) using activator (DNFB))
A conjugate of 2Me-2OH5N_C (Example 11, FIG. 3) was produced using DNFB in the following manner. Nucleic acid strand 2Meps containing 3'-phosphorothioate modification (Fig. 3) (50 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Fig. 3) (10 pmol), and template nucleic acid strand tmr27-II ( Figure 3) (20 pmol) was dissolved in a solution (total volume 18 μL) containing 50 mM sodium phosphate and 100 mM sodium chloride (pH 8). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Add 2 μL of DMSO solution containing 100 mM DNFB to reaction solution 1, react in a total volume of 20 μL at 27°C for 6 hours, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to prepare a solution containing the conjugate. I got it. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). .
　（活性化剤（ＤＮＦＢ）を利用したＲＮＡの連結体（２Ｍｅ―２Ｆ５Ｎ＿Ｃ）（実施例１２）の作製）
　　　ＤＮＦＢを利用して連結体の２Ｍｅ―２Ｆ５Ｎ＿Ｃ（実施例１２、図３）を以下の方法で作製した。３’－ホスホロチオエート修飾を含む核酸鎖２Ｍｅｐｓ（図３）（５０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２Ｆ５Ｎ＿Ｃ（図３）（１０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ２７－ＩＩ（２０ｐｍｏｌ）とを５０ｍＭリン酸ナトリウム、１００ｍＭ塩化ナトリウム（ｐＨ８）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。１００ｍＭ　ＤＮＦＢを含むＤＭＳＯ溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６時間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。 (Preparation of RNA conjugate (2Me-2F5N_C) (Example 12) using activator (DNFB))
A conjugate, 2Me-2F5N_C (Example 12, FIG. 3), was produced by the following method using DNFB. Nucleic acid strand 2Meps containing 3'-phosphorothioate modification (Fig. 3) (50 pmol), nucleic acid strand 2F5N_C containing 5' amino modification and modified with a fluorescent molecule (Fig. 3) (10 pmol), and template nucleic acid strand tmr27-II ( 20 pmol) was dissolved in a solution (total volume 18 μL) containing 50 mM sodium phosphate and 100 mM sodium chloride (pH 8). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). Add 2 μL of DMSO solution containing 100 mM DNFB to reaction solution 1, react in a total volume of 20 μL at 27°C for 6 hours, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to prepare a solution containing the conjugate. I got it. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). .
（ＤＮＡテンプレート及び活性化剤（ＤＮＦＢ）を利用したＲＮＡの連結体（実施例１３）の作製）
ＤＮＦＢを活性化剤として利用して、上記２Ｍｅ―２ＯＨ５Ｎ＿Ｃ、および２Ｍｅ―２Ｆ５Ｎ＿Ｃを、ＤＮＡテンプレートｔｍｄ２７－ＩＩを用いて同様に作製した。図１４に示すように、ＲＮＡテンプレートと同様に２Ｍｅ―２ＯＨ５Ｎ＿Ｃと比較して２Ｍｅ―２Ｆ５Ｎ＿Ｃは高い反応収量を示したことから、２Ｆ５Ｎ＿ＣはＤＮＦＢを用いた連結反応において２ＯＨ５Ｎ＿Ｃよりも有効であることを確認した。 (Preparation of RNA conjugate (Example 13) using DNA template and activator (DNFB))
Using DNFB as an activator, the above 2Me-2OH5N_C and 2Me-2F5N_C were similarly produced using DNA template tmd27-II. As shown in Figure 14, similar to the RNA template, 2Me-2F5N_C showed a higher reaction yield compared to 2Me-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction using DNFB. did.
　（５’ｏｌｉｇｏとテンプレートＤＮＡ及びＲＮＡのＴｍ測定）
　３’－ホスホロチオエート修飾を含む核酸鎖２Ｍｅｐｓおよび２Ｆｐｓ（図３）、についてテンプレート核酸との融解曲線を測定した。標的となる核酸として、ＤＮＡのｔｍｒ２７（図３）及びＲＮＡのｔｍｄ２７（図３）を選択した。 (Tm measurement of 5'oligo and template DNA and RNA)
Melting curves with the template nucleic acid were measured for nucleic acid strands 2Meps and 2Fps (FIG. 3) containing 3'-phosphorothioate modification. DNA tmr27 (FIG. 3) and RNA tmd27 (FIG. 3) were selected as target nucleic acids.
　（３’ｏｌｉｇｏとテンプレートＤＮＡ及びＲＮＡのＴｍ測定）　　
　５’アミノ修飾を含む核酸鎖２ＯＨ５Ｎ、および２Ｆ５Ｎについて、テンプレート核酸との融解曲線を測定した。標的となる核酸として、ＤＮＡのｔｍｒ２７（図３）及びＲＮＡのｔｍｄ２７（図３）を選択した。 (Tm measurement of 3'oligo and template DNA and RNA)
Melting curves of nucleic acid chains 2OH5N and 2F5N containing 5' amino modification with the template nucleic acid were measured. DNA tmr27 (FIG. 3) and RNA tmd27 (FIG. 3) were selected as target nucleic acids.
　表１に示すように、３’－ホスホロチオエート修飾を含む核酸鎖２Ｆｐｓでは、ＲＮＡテンプレートおよびＤＮＡテンプレートどちらにおいても２Ｍｅｐｓに比して、高いＴｍ値をしめした。また、５’アミノ修飾を含む２Ｆ５Ｎでは、ＲＮＡテンプレートおよびＤＮＡテンプレートどちらにおいても２ＯＨ５Ｎに比して高いＴｍ値をしめした。
表１．5’-oligo、または3’-oligoのテンプレート核酸とのTm解析
As shown in Table 1, the nucleic acid strand 2Fps containing the 3'-phosphorothioate modification showed a higher Tm value than 2Meps in both the RNA template and the DNA template. Furthermore, 2F5N containing 5' amino modification showed a higher Tm value than 2OH5N in both RNA template and DNA template.
Table 1. Tm analysis of 5'-oligo or 3'-oligo with template nucleic acid
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｍｅ―２ＯＨ５Ｎ）（実施例１４）の作製）
　ＥＤＣを利用して連結体の２Ｍｅ―２ＯＨ５Ｎ（実施例１４、図３）を以下の方法で作製した。核酸鎖２Ｍｅｐｏ（図３）（２０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（２０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ１２（図３）（２０ｐｍｏｌ）とを２５０ｍＭ　ＨＥＰＥＳ、１Ｍ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６０分間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、蛍光イメージングを用いて分析した。より具体的には、実施例１４の連結体は、原料である蛍光分子で修飾された核酸鎖２ＯＨ５Ｎの核酸鎖よりも遅れて移動することが確認された。ゲルの分析は電気泳動後にＴｙｐｈｏｏｎ　ＦＬＡ９０００（ＧＥ　Ｈｅａｌｔｈ．）によって解析した。 (Preparation of RNA conjugate (2Me-2OH5N) (Example 14) using carbodiimide-based activator (EDC))
Using EDC, the conjugate 2Me-2OH5N (Example 14, FIG. 3) was produced by the following method. Nucleic acid strand 2Mepo (Fig. 3) (20 pmol), nucleic acid strand 2OH5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 μL) containing HEPES and 1M sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 μL of a solution containing HCl to reaction solution 1, react in a total volume of 20 μL at 27°C for 60 minutes, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging. More specifically, it was confirmed that the conjugate of Example 14 migrated later than the 2OH5N nucleic acid chain modified with a fluorescent molecule as a raw material. Gel analysis was performed using Typhoon FLA9000 (GE Health.) after electrophoresis.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｍｅ―２Ｆ５Ｎ）（実施例１５）の作製）
　ＥＤＣを利用して連結体の２Ｍｅ―２Ｆ５Ｎ（実施例１５、図３）を以下の方法で作製した。核酸鎖２Ｍｅｐｏ（図３）（２０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２Ｆ５Ｎ（図３）（２０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ１２（図３）（２０ｐｍｏｌ）とを２５０ｍＭ　ＨＥＰＥＳ、１Ｍ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６０分間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。図１０Ａに示すように、２Ｍｅ―２ＯＨ５Ｎと比較して２Ｍｅ―２Ｆ５Ｎは高い反応収量を示したことから、２Ｆ５Ｎはカルボジイミドを用いた核酸鎖２Ｍｅｐｏとの連結反応でも２ＯＨ５Ｎよりも有効であることを確認した。 (Preparation of RNA conjugate (2Me-2F5N) (Example 15) using carbodiimide-based activator (EDC))
A conjugate of 2Me-2F5N (Example 15, FIG. 3) was produced by the following method using EDC. Nucleic acid strand 2Mepo (Fig. 3) (20 pmol), nucleic acid strand 2F5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 μL) containing HEPES and 1M sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 μL of a solution containing HCl to reaction solution 1, react in a total volume of 20 μL at 27°C for 60 minutes, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). As shown in Figure 10A, 2Me-2F5N showed a higher reaction yield compared to 2Me-2OH5N, confirming that 2F5N is more effective than 2OH5N in the ligation reaction with the nucleic acid chain 2Mepo using carbodiimide. did.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｆ―２ＯＨ５Ｎ）（実施例１６）の作製）
　ＥＤＣを利用して連結体の２Ｆ―２ＯＨ５Ｎ（実施例１６、図３）を以下の方法で作製した。核酸鎖２Ｆｐｏ（図３）（２０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（２０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ１２（図３）（２０ｐｍｏｌ）とを２５０ｍＭ　ＨＥＰＥＳ、１Ｍ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６０分間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。 (Preparation of RNA conjugate (2F-2OH5N) (Example 16) using carbodiimide-based activator (EDC))
A conjugate of 2F-2OH5N (Example 16, FIG. 3) was prepared using EDC in the following manner. Nucleic acid strand 2Fpo (Fig. 3) (20 pmol), nucleic acid strand 2OH5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 μL) containing HEPES and 1M sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 μL of a solution containing HCl to reaction solution 1, react in a total volume of 20 μL at 27°C for 60 minutes, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｆ―２Ｆ５Ｎ）（実施例１７）の作製）
　ＥＤＣを利用して連結体の２Ｆ―２Ｆ５Ｎ（実施例１７、図３）を以下の方法で作製した。核酸鎖２Ｆｐｏ（図３）（２０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２Ｆ５Ｎ（図３）（２０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ１２（図３）（２０ｐｍｏｌ）とを２５０ｍＭ　ＨＥＰＥＳ、１Ｍ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６０分間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。図１０Ａに示すように、２Ｆ―２ＯＨ５Ｎと比較して２Ｆ―２Ｆ５Ｎは高い反応収量を示したことから、２Ｆ５Ｎはカルボジイミドを用いた核酸鎖２Ｆｐｏとの連結反応でも２ＯＨ５Ｎよりも有効であることを確認した。 (Preparation of RNA conjugate (2F-2F5N) (Example 17) using carbodiimide-based activator (EDC))
Using EDC, the conjugate 2F-2F5N (Example 17, FIG. 3) was produced by the following method. Nucleic acid strand 2Fpo (Fig. 3) (20 pmol), nucleic acid strand 2F5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 μL) containing HEPES and 1M sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 μL of a solution containing HCl to reaction solution 1, react in a total volume of 20 μL at 27°C for 60 minutes, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). As shown in Figure 10A, 2F-2F5N showed a higher reaction yield compared to 2F-2OH5N, confirming that 2F5N is more effective than 2OH5N in the ligation reaction with the nucleic acid chain 2Fpo using carbodiimide. did.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｈ―２ＯＨ５Ｎ）（実施例１８）の作製）
　ＥＤＣを利用して連結体の２Ｈ―２ＯＨ５Ｎ（実施例１８、図３）を以下の方法で作製した。核酸鎖２Ｈｐｏ（図３）（２０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ（図３）（２０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ１２（図３）（２０ｐｍｏｌ）とを２５０ｍＭ　ＨＥＰＥＳ、１Ｍ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６０分間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例3）と同様の方法で蛍光イメージングを用いて解析した。 (Preparation of RNA conjugate (2H-2OH5N) (Example 18) using carbodiimide-based activator (EDC))
Using EDC, the conjugate 2H-2OH5N (Example 18, FIG. 3) was produced by the following method. Nucleic acid strand 2Hpo (Fig. 3) (20 pmol), nucleic acid strand 2OH5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 μL) containing HEPES and 1M sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 μL of a solution containing HCl to reaction solution 1, react in a total volume of 20 μL at 27°C for 60 minutes, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｈ―２Ｆ５Ｎ）（実施例１９）の作製）
　ＥＤＣを利用して連結体の２Ｈ―２Ｆ５Ｎ（実施例１９、図３）を以下の方法で作製した。核酸鎖２Ｈｐｏ（図３）（２０ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２Ｆ５Ｎ（図３）（２０ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ１２（図３）（２０ｐｍｏｌ）とを２５０ｍＭ　ＨＥＰＥＳ、１Ｍ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量１８μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を２μＬ反応液１に加え、総量２０μＬで２７℃、６０分間反応を行った後、３μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ　（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１２μＬと混合して連結体を含む溶液を得た。その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。図１０Ａに示すように、２Ｈ―２ＯＨ５Ｎと比較して２Ｈ―２Ｆ５Ｎは高い反応収量を示したことから、２Ｆ５Ｎはカルボジイミドを用いた核酸鎖２Ｈｐｏとの連結反応でも２ＯＨ５Ｎよりも有効であることを確認した。 (Preparation of RNA conjugate (2H-2F5N) (Example 19) using carbodiimide-based activator (EDC))
Using EDC, the conjugate 2H-2F5N (Example 19, FIG. 3) was produced by the following method. Nucleic acid strand 2Hpo (Fig. 3) (20 pmol), nucleic acid strand 2F5N (Fig. 3) (20 pmol) containing 5' amino modification and modified with a fluorescent molecule, and template nucleic acid strand tmr12 (Fig. 3) (20 pmol) at 250 mM. It was dissolved in a solution (total volume 18 μL) containing HEPES and 1M sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 2 μL of a solution containing HCl to reaction solution 1, react in a total volume of 20 μL at 27°C for 60 minutes, and then mix 3 μL with 12 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). As shown in Figure 10A, 2H-2F5N showed a higher reaction yield compared to 2H-2OH5N, confirming that 2F5N is more effective than 2OH5N in the ligation reaction with the nucleic acid chain 2Hpo using carbodiimide. did.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｍｅ―２ＯＨ５Ｎ）（実施例２０）の作製）
　カルボジイミド系の活性化剤（ＥＤＣ）を利用して連結体の２Ｍｅ―２ＯＨ５Ｎ（実施例２０、図３）を以下の方法で作製した。核酸鎖２Ｍｅｐｏ（図３）（５００ｐｍｏｌ）、５’アミノ修飾を含む核酸鎖２ＯＨ５Ｎ（図３）（５００ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｒ１２（図３）（５００ｐｍｏｌ）とを２５０ｍＭ　ＨＥＰＥＳ、１Ｍ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量９０μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を１０μＬ反応液１に加え、総量１００μＬで１７℃、６時間反応を行った後、ゲルろ過カラム（ＧＥ　Ｈｅａｌｔｈｃａｒｅ、ＮＡＰ－５）を行って粗精製した。その後、逆相カラムを用いたＨＰＬＣを用いて精製し、２Ｍｅ―２ＯＨ５Ｎを得た。精製には下記の溶液を用いた。
　Ａ溶液：５％アセトニトリル／０．１Ｍ　ＴＥＡＡ（ｐＨ７．０）
　Ｂ溶液：２５％アセトニトリル／０．１Ｍ　ＴＥＡＡ（ｐＨ７．０）
より具体的には図１０Ｖに示すように、実施例２０のＲＮＡの連結体２Ｍｅ―２ＯＨ５Ｎは、原料である２Ｍｅｐｏおよび２ＯＨ５Ｎよりも遅れてピークが観察されることが確認された。 (Preparation of RNA conjugate (2Me-2OH5N) (Example 20) using carbodiimide-based activator (EDC))
A conjugate of 2Me-2OH5N (Example 20, FIG. 3) was produced by the following method using a carbodiimide-based activator (EDC). Nucleic acid strand 2Mepo (Fig. 3) (500 pmol), nucleic acid strand 2OH5N containing 5' amino modification (Fig. 3) (500 pmol), and template nucleic acid strand tmr12 (Fig. 3) (500 pmol) were mixed in 250 mM HEPES, 1 M sodium chloride ( pH 7.3) (total volume: 90 μL). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. A solution containing HCl was added to 10 μL of reaction solution 1, and the reaction was carried out at 17° C. for 6 hours in a total volume of 100 μL, followed by rough purification using a gel filtration column (GE Healthcare, NAP-5). Thereafter, it was purified using HPLC using a reverse phase column to obtain 2Me-2OH5N. The following solution was used for purification.
Solution A: 5% acetonitrile/0.1M TEAA (pH 7.0)
Solution B: 25% acetonitrile/0.1M TEAA (pH 7.0)
More specifically, as shown in FIG. 10V, it was confirmed that the peak of the RNA linkage 2Me-2OH5N of Example 20 was observed later than that of the raw materials 2Mepo and 2OH5N.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｍｅ―２Ｆ５Ｎ）（実施例２１）の作製）
　カルボジイミド系の活性化剤（ＥＤＣ）を利用して連結体の２Ｍｅ―２Ｆ５Ｎ（実施例２１、図３）を上記２Ｍｅ―２ＯＨ５Ｎと同様の方法で作製した。より具体的には図１０Ｃに示すように、実施例２１のＲＮＡの連結体２Ｍｅ―２Ｆ５Ｎは、原料である２Ｍｅｐｏおよび２Ｆ５Ｎよりも遅れてピークが観察されることが確認された。 (Preparation of RNA conjugate (2Me-2F5N) (Example 21) using carbodiimide-based activator (EDC))
A conjugate of 2Me-2F5N (Example 21, FIG. 3) was prepared using a carbodiimide-based activator (EDC) in the same manner as for the above-mentioned 2Me-2OH5N. More specifically, as shown in FIG. 10C, it was confirmed that the peak of the RNA linkage 2Me-2F5N of Example 21 was observed later than that of the raw materials 2Mepo and 2F5N.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｍｅ―２ＯＨ５Ｎ_Ｃ）（実施例２２）の作製）
　ＥＤＣを利用して連結体の２Ｍｅ―２ＯＨ５Ｎ_Ｃ（実施例２２、図３）を以下の方法で作製した。核酸鎖２Ｍｅｐｏ（図３）（３ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ_Ｃ（図３）（３ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｄ２７－ＩＩ（図３）（３ｐｍｏｌ）とを５０ｍＭ　ＨＥＰＥＳ、１０ｍＭ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量２７μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を３μＬ反応液１に加え、総量３０μＬで３７℃、３０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１５μＬと混合して連結体を含む溶液を得た。その後、１５％アクリルアミド、２５％ホルムアミド、５．６Ｍ　尿素、１ｘＴＢＥを含むポリアクリルアミドゲルにアプライして電気泳動し、蛍光イメージングを用いて分析した。より具体的には、実施例２２の連結体は、原料である蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ_Ｃの核酸鎖よりも遅れて移動することが確認された。ゲルの分析は電気泳動後にＴｙｐｈｏｏｎ　ＦＬＡ９０００（ＧＥ　Ｈｅａｌｔｈ．）によって解析した。 (Preparation of RNA conjugate (2Me-2OH5N_C) (Example 22) using carbodiimide-based activator (EDC))
A conjugate of 2Me-2OH5N_C (Example 22, FIG. 3) was produced using EDC in the following manner. Nucleic acid strand 2Mepo (Figure 3) (3 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Figure 3) (3 pmol), and template nucleic acid strand tmd27-II (Figure 3) (3 pmol). was dissolved in a solution (total volume 27 μL) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 μL of a solution containing HCl to reaction solution 1, react in a total volume of 30 μL at 37°C for 30 minutes, and then mix 5 μL with 15 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was applied to a polyacrylamide gel containing 15% acrylamide, 25% formamide, 5.6M urea, and 1x TBE, subjected to electrophoresis, and analyzed using fluorescence imaging. More specifically, it was confirmed that the conjugate of Example 22 migrated later than the nucleic acid strand of the fluorescent molecule-modified nucleic acid strand 2OH5N_C, which was the raw material. Gel analysis was performed using Typhoon FLA9000 (GE Health.) after electrophoresis.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｍｅ―２Ｆ５Ｎ_Ｃ）（実施例２３）の作製）
　ＥＤＣを利用して連結体の２Ｍｅ―２Ｆ５Ｎ_Ｃ（実施例２３、図３）を以下の方法で作製した。核酸鎖２Ｍｅｐｏ（図３）（３ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ_Ｃ（図３）（３ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｄ２７－ＩＩ（図３）（３ｐｍｏｌ）とを５０ｍＭ　ＨＥＰＥＳ、１０ｍＭ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量２７μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を３μＬ反応液１に加え、総量３０μＬで３７℃、３０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１５μＬと混合して連結体を含む溶液を得た。その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。図１５に示すように、２Ｍｅ―２ＯＨ５Ｎ_Ｃと比較して２Ｍｅ―２Ｆ５Ｎ_Ｃは高い反応収量を示したことから、２Ｆ５Ｎ_Ｃはカルボジイミドを用いた核酸鎖２Ｍｅｐｏとの連結反応でも２ＯＨ５Ｎ_Ｃよりも有効であることを確認した。 (Preparation of RNA conjugate (2Me-2F5N_C) (Example 23) using carbodiimide-based activator (EDC))
A conjugate of 2Me-2F5N_C (Example 23, FIG. 3) was produced using EDC in the following manner. Nucleic acid strand 2Mepo (Figure 3) (3 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Figure 3) (3 pmol), and template nucleic acid strand tmd27-II (Figure 3) (3 pmol). was dissolved in a solution (total volume 27 μL) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 μL of a solution containing HCl to reaction solution 1, react in a total volume of 30 μL at 37°C for 30 minutes, and then mix 5 μL with 15 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). As shown in Figure 15, 2Me-2F5N_C showed a higher reaction yield compared to 2Me-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction with the nucleic acid chain 2Mepo using carbodiimide. did.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｆ―２ＯＨ５Ｎ_Ｃ）（実施例２４）の作製）
　ＥＤＣを利用して連結体の２Ｆ―２ＯＨ５Ｎ_Ｃ（実施例２４、図３）を以下の方法で作製した。核酸鎖２Ｆｐｏ（図３）（３ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ_Ｃ（図３）（３ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｄ２７－ＩＩ（図３）（３ｐｍｏｌ）とを５０ｍＭ　ＨＥＰＥＳ、１０ｍＭ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量２７μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を３μＬ反応液１に加え、総量３０μＬで３７℃、３０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１５μＬと混合して連結体を含む溶液を得た。その後、その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。 (Preparation of RNA conjugate (2F-2OH5N_C) (Example 24) using carbodiimide-based activator (EDC))
A conjugate, 2F-2OH5N_C (Example 24, FIG. 3), was produced using EDC in the following manner. Nucleic acid strand 2Fpo (Fig. 3) (3 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Fig. 3) (3 pmol), and template nucleic acid strand tmd27-II (Fig. 3) (3 pmol). was dissolved in a solution (total volume 27 μL) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 μL of a solution containing HCl to reaction solution 1, react in a total volume of 30 μL at 37°C for 30 minutes, and then mix 5 μL with 15 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｆ―２Ｆ５Ｎ_Ｃ）（実施例２５）の作製）
　　　ＥＤＣを利用して連結体の２Ｆ―２Ｆ５Ｎ_Ｃ（実施例２５、図３）を以下の方法で作製した。核酸鎖２Ｆｐｏ（図３）（３ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ_Ｃ（図３）（３ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｄ２７－ＩＩ（図３）（３ｐｍｏｌ）とを５０ｍＭ　ＨＥＰＥＳ、１０ｍＭ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量２７μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を３μＬ反応液１に加え、総量３０μＬで３７℃、３０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１５μＬと混合して連結体を含む溶液を得た。その後、その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。図１５に示すように、２Ｆ―２ＯＨ５Ｎ_Ｃと比較して２Ｆ―２Ｆ５Ｎ_Ｃは高い反応収量を示したことから、２Ｆ５Ｎ_Ｃはカルボジイミドを用いた核酸鎖２Ｆｐｏとの連結反応でも２ＯＨ５Ｎ_Ｃよりも有効であることを確認した。 (Preparation of RNA conjugate (2F-2F5N_C) (Example 25) using carbodiimide-based activator (EDC))
A conjugate, 2F-2F5N_C (Example 25, FIG. 3), was produced by the following method using EDC. Nucleic acid strand 2Fpo (Figure 3) (3 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Figure 3) (3 pmol), and template nucleic acid strand tmd27-II (Figure 3) (3 pmol). was dissolved in a solution (total volume 27 μL) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 μL of a solution containing HCl to reaction solution 1, react in a total volume of 30 μL at 37°C for 30 minutes, and then mix 5 μL with 15 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). As shown in Figure 15, 2F-2F5N_C showed a higher reaction yield compared to 2F-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction with the nucleic acid chain 2Fpo using carbodiimide. did.
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｈ―２ＯＨ５Ｎ_Ｃ）（実施例２６）の作製）
　ＥＤＣを利用して連結体の２Ｈ―２ＯＨ５Ｎ_Ｃ（実施例２６、図３）を以下の方法で作製した。核酸鎖２Ｈｐｏ（図３）（３ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ_Ｃ（図３）（３ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｄ２７－ＩＩ（図３）（３ｐｍｏｌ）とを５０ｍＭ　ＨＥＰＥＳ、１０ｍＭ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量２７μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を３μＬ反応液１に加え、総量３０μＬで３７℃、３０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１５μＬと混合して連結体を含む溶液を得た。その後、その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。 (Preparation of RNA conjugate (2H-2OH5N_C) (Example 26) using carbodiimide-based activator (EDC))
Using EDC, the conjugate 2H-2OH5N_C (Example 26, FIG. 3) was produced by the following method. Nucleic acid strand 2Hpo (Figure 3) (3 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Figure 3) (3 pmol), and template nucleic acid strand tmd27-II (Figure 3) (3 pmol). was dissolved in a solution (total volume 27 μL) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 μL of a solution containing HCl to reaction solution 1, react in a total volume of 30 μL at 37°C for 30 minutes, and then mix 5 μL with 15 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3).
　（カルボジイミド系の活性化剤（ＥＤＣ）を利用したＲＮＡの連結体（２Ｆ―２Ｆ５Ｎ_Ｃ）（実施例２７）の作製）
　ＥＤＣを利用して連結体の２Ｆ―２Ｆ５Ｎ_Ｃ（実施例２７、図３）を以下の方法で作製した。核酸鎖２Ｈｐｏ（図３）（３ｐｍｏｌ）、５’アミノ修飾を含み蛍光分子で修飾された核酸鎖２ＯＨ５Ｎ_Ｃ（図３）（３ｐｍｏｌ）とテンプレートとなる核酸鎖ｔｍｄ２７－ＩＩ（図３）（３ｐｍｏｌ）とを５０ｍＭ　ＨＥＰＥＳ、１０ｍＭ塩化ナトリウム（ｐＨ７．３）を含む溶液（総量２７μＬ）に溶解させた。反応液を９０℃で１分加熱し氷冷して３分間放置した（反応液１）。６Ｍ　ＥＤＣ．ＨＣｌを含む溶液を３μＬ反応液１に加え、総量３０μＬで３７℃、３０分間反応を行った後、５μＬをｌｏａｄｉｎｇ　ｂｕｆｆｅｒ（８０％ホルムアミド、１０ｍＭ　ＥＤＴＡ）１５μＬと混合して連結体を含む溶液を得た。その後、その後、上記２Ｍｅ―２ＯＨ５Ｎ（実施例３）と同様の方法で蛍光イメージングを用いて解析した。図１５に示すように、２Ｈ―２ＯＨ５Ｎ_Ｃと比較して２Ｈ―２Ｆ５Ｎ_Ｃは高い反応収量を示したことから、２Ｆ５Ｎ_Ｃはカルボジイミドを用いた核酸鎖２Ｈｐｏとの連結反応でも２ＯＨ５Ｎ_Ｃよりも有効であることを確認した。 (Preparation of RNA conjugate (2F-2F5N_C) (Example 27) using carbodiimide-based activator (EDC))
A conjugate, 2F-2F5N_C (Example 27, FIG. 3), was produced by the following method using EDC. Nucleic acid strand 2Hpo (Figure 3) (3 pmol), nucleic acid strand 2OH5N_C containing 5' amino modification and modified with a fluorescent molecule (Figure 3) (3 pmol), and template nucleic acid strand tmd27-II (Figure 3) (3 pmol). was dissolved in a solution (total volume 27 μL) containing 50 mM HEPES and 10 mM sodium chloride (pH 7.3). The reaction solution was heated at 90° C. for 1 minute, cooled on ice, and left for 3 minutes (Reaction Solution 1). 6M EDC. Add 3 μL of a solution containing HCl to reaction solution 1, react in a total volume of 30 μL at 37°C for 30 minutes, and then mix 5 μL with 15 μL of loading buffer (80% formamide, 10 mM EDTA) to obtain a solution containing the conjugate. Ta. Thereafter, it was analyzed using fluorescence imaging in the same manner as the above 2Me-2OH5N (Example 3). As shown in Figure 15, 2H-2F5N_C showed a higher reaction yield compared to 2H-2OH5N_C, confirming that 2F5N_C is more effective than 2OH5N_C in the ligation reaction with nucleic acid chain 2Hpo using carbodiimide. did.
　（実施例２８）
　３’位がリン酸基修飾されているオリゴヌクレオチドまたはその改変体（第一のオリゴヌクレオチドまたはその改変体）の５’末端と、５’位にアミノ修飾されているオリゴヌクレオチドまたはその改変体（第二のオリゴヌクレオチドまたはその改変体）の３’末端とが連結したオリゴヌクレオチドまたはその改変体の複数と、３’位がリン酸基修飾されているオリゴヌクレオチドまたはその改変体（第一のオリゴヌクレオチドまたはその改変体）と、５’位にアミノ修飾されているオリゴヌクレオチドまたはその改変体（第二のオリゴヌクレオチドまたはその改変体）とを、活性化剤の存在下で反応さして、2か所以上同時にまたは段階的に連結し、複数のオリゴヌクレオチドまたはその改変体が連結したオリゴヌクレオチドまたはその改変体を作製する。
(Example 28)
The 5' end of the oligonucleotide or its variant (the first oligonucleotide or its variant) whose 3' position is modified with a phosphate group, and the oligonucleotide or its variant whose 5' position is amino-modified (the first oligonucleotide or its variant). A plurality of oligonucleotides or variants thereof linked to the 3' end of a second oligonucleotide or a variant thereof) and an oligonucleotide or a variant thereof whose 3' position is modified with a phosphate group (the first oligonucleotide or a variant thereof) A nucleotide or a variant thereof) is reacted with an oligonucleotide or a variant thereof that is amino-modified at the 5' position (a second oligonucleotide or a variant thereof) in the presence of an activator. The above is linked simultaneously or stepwise to produce an oligonucleotide or a variant thereof in which a plurality of oligonucleotides or variants thereof are linked.
　（実施例２９）
　テンプレート核酸が５’位にアミノ修飾されているオリゴヌクレオチドまたはその改変体（第二のオリゴヌクレオチドまたはその改変体）の３’末端に連結しているオリゴヌクレオチドまたはその改変体と、３’位がリン酸基修飾されているオリゴヌクレオチドまたはその改変体（第一のオリゴヌクレオチドまたはその改変体）を、活性化剤の存在下で反応させて、連結されたオリゴヌクレオチドまたはその改変体を作製する。
(Example 29)
An oligonucleotide or a variant thereof in which the template nucleic acid is amino-modified at the 5' position or a variant thereof (a second oligonucleotide or a variant thereof), and an oligonucleotide or a variant thereof that is amino-modified at the 5' position. A phosphate group-modified oligonucleotide or a variant thereof (first oligonucleotide or a variant thereof) is reacted in the presence of an activator to produce a linked oligonucleotide or a variant thereof.
　（実施例３０）
　テンプレート核酸が、３’位がリン酸基修飾されているオリゴヌクレオチドまたはその改変体（第一のオリゴヌクレオチドまたはその改変体）の５’末端に連結しているオリゴヌクレオチドまたはその改変体と、５’位にアミノ修飾されているオリゴヌクレオチドまたはその改変体（第二のオリゴヌクレオチドまたはその改変体）とを、活性化剤の存在下で反応させて、連結されたオリゴヌクレオチドまたはその改変体を作製する。
(Example 30)
An oligonucleotide or a variant thereof in which the template nucleic acid is linked to the 5' end of an oligonucleotide or a variant thereof (first oligonucleotide or a variant thereof) whose 3' position is modified with a phosphate group; A linked oligonucleotide or a variant thereof is produced by reacting an oligonucleotide amino-modified at the ' position or a variant thereof (a second oligonucleotide or a variant thereof) in the presence of an activating agent. do.
　（実施例３１）
　テンプレート核酸の両端が、３’位がリン酸基修飾されているオリゴヌクレオチドまたはその改変体（第一のオリゴヌクレオチドまたはその改変体）の５’末端に連結し、５’位にアミノ修飾されているオリゴヌクレオチドまたはその改変体（第二のオリゴヌクレオチドまたはその改変体）の３’末端に連結している、オリゴヌクレオチドまたはその改変体を、活性化剤の存在下で反応させて、オリゴヌクレオチドまたはその改変体の３’末端と５’末端とが連結した環状オリゴヌクレオチドまたはその改変体を作製する。
(Example 31)
Both ends of the template nucleic acid are linked to the 5' end of an oligonucleotide or a variant thereof (the first oligonucleotide or a variant thereof) whose 3' position is modified with a phosphate group, and the 5' position is amino-modified. The oligonucleotide or its variant linked to the 3' end of the oligonucleotide or its variant (second oligonucleotide or its variant) is reacted in the presence of an activator to form the oligonucleotide or its variant. A cyclic oligonucleotide or a variant thereof in which the 3' end and 5' end of the variant are linked is produced.
　（実施例３２：製剤例）
　本開示の化合物は、適宜の剤形にて提供され得る。以下にその例を示す。
　錠剤、カプセル剤、散剤、顆粒剤、液剤、懸濁剤、注射剤、貼付剤、パップ剤等が挙げられる。また、これらの製剤は、通常の医薬品添加物として使用されている添加剤を用いて、これらを適宜混合などを行うことを含む公知の方法で製造することができる。 (Example 32: Formulation example)
Compounds of the present disclosure may be provided in any suitable dosage form. An example is shown below.
Examples include tablets, capsules, powders, granules, solutions, suspensions, injections, patches, poultices, and the like. Further, these preparations can be manufactured by a known method using additives commonly used as pharmaceutical additives, including appropriately mixing them.
　（実施例３３：動物実験）
　疾患を有するマウス（例えば、担癌マウス）を用い、本開示のオリゴヌクレオチドまたは偽薬を腹腔内に投与する。
　その後、偽薬とオリゴヌクレオチドとの比較を行い、本開示のオリゴヌクレオチドのマウス体内における疾患に対する有効性を評価することができる。 (Example 33: Animal experiment)
A diseased mouse (eg, a tumor-bearing mouse) is used, and the oligonucleotide of the present disclosure or a placebo is administered intraperitoneally.
Thereafter, a comparison between a placebo and the oligonucleotide can be performed to evaluate the effectiveness of the oligonucleotide of the present disclosure against diseases in the mouse body.
　（実施例３４）
T.　Seya　et　al.　Advanced　Drug　Delivery　Reviews　147　2019　37-43に掲載されているような比較的長めの2本鎖を、テンプレートのDNAを用いずに、お互いの鎖がテンプレートになるように作製することも想定され得る（図１１）。実施例３と同様の方法で、第一のオリゴヌクレオチドまたはその改変体および第二のオリゴヌクレオチドまたはその改変体を用いて、活性化剤の存在下で反応させて、連結されたオリゴヌクレオチドまたはその改変体を作製する。 (Example 34)
Create a relatively long double strand as described in T. Seya et al. Advanced Drug Delivery Reviews 147 2019 37-43 without using template DNA so that each strand serves as a template It can also be assumed (FIG. 11). In the same manner as in Example 3, a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof are reacted in the presence of an activator to form a linked oligonucleotide or its variant. Create a variant.
　（注記）
　以上のように、本開示の好ましい実施形態を用いて本開示を例示してきたが、本開示は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願および他の文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。 (Note)
As described above, although the present disclosure has been illustrated using the preferred embodiments of the present disclosure, it is understood that the scope of the present disclosure should be interpreted only by the claims. The patents, patent applications, and other documents cited herein are hereby incorporated by reference to the same extent as if the contents themselves were specifically set forth herein. That is understood.
　本開示で提供される技術は、オリゴヌクレオチドまたはその改変体を連結させる技術を利用するあらゆる分野において利用することができる。 The technology provided in the present disclosure can be used in any field that uses technology for linking oligonucleotides or variants thereof.
配列番号１：RNA　template　(tmr27)
配列番号２：DNA　template　(tmd27)
配列番号３：RNA　template　(tmr12)
配列番号４：第一のオリゴヌクレオチド：5’oligo（cytidine）
配列番号５：第一のオリゴヌクレオチド：5’oligo（2’O-methyl-cytidine）
配列番号６：第二のオリゴヌクレオチド：3’oligo（2’H-　Uridine）
配列番号７：第一のオリゴヌクレオチド+テンプレート核酸、tmr12+5’oligo（cytidine）
配列番号８：テンプレート核酸+第一のオリゴヌクレオチド、tmr12+5’oligo（2’O-methyl-cytidine）
配列番号９：第二のオリゴヌクレオチド+テンプレート核酸、3’oligo（2’H-Uridine）+tmr12
配列番号１０：第二のオリゴヌクレオチド+テンプレート核酸+第一のオリゴヌクレオチド：3’oligo（2’H-Uridine）+tmr12+5’oligo（cytidine）
配列番号１１：第二のオリゴヌクレオチド+第一のオリゴヌクレオチド：3’oligo（2’H-Uridine）+5’oligo（2’O-methyl-cytidine）
配列番号１２：RNA　template(実施例28)
配列番号１３：第二のオリゴヌクレオチド+第一のオリゴヌクレオチド、3’oligo（2’H-Uridine）+5’oligo（cytidine）(実施例28)
配列番号１４：第二のオリゴヌクレオチド+第一のオリゴヌクレオチド、3’oligo（2’H-Uridine）+5’oligo（2’O-methyl-cytidine）(実施例28)
配列番号１５：第二のオリゴヌクレオチド、3’oligo（2’H-Uridine）(実施例28)
配列番号１６：RNA　template　(tmr27-II)
配列番号１７：DNA　template　(tmd27-II) Sequence number 1: RNA template (tmr27)
Sequence number 2: DNA template (tmd27)
Sequence number 3: RNA template (tmr12)
SEQ ID NO: 4: First oligonucleotide: 5'oligo (cytidine)
SEQ ID NO: 5: First oligonucleotide: 5'oligo (2'O-methyl-cytidine)
SEQ ID NO: 6: Second oligonucleotide: 3'oligo (2'H-Uridine)
SEQ ID NO: 7: First oligonucleotide + template nucleic acid, tmr12+5'oligo (cytidine)
SEQ ID NO: 8: Template nucleic acid + first oligonucleotide, tmr12 + 5'oligo (2'O-methyl-cytidine)
SEQ ID NO: 9: Second oligonucleotide + template nucleic acid, 3'oligo (2'H-Uridine) + tmr12
SEQ ID NO: 10: Second oligonucleotide + template nucleic acid + first oligonucleotide: 3'oligo (2'H-Uridine) + tmr12 + 5'oligo (cytidine)
SEQ ID NO: 11: Second oligonucleotide + first oligonucleotide: 3'oligo (2'H-Uridine) + 5'oligo (2'O-methyl-cytidine)
SEQ ID NO: 12: RNA template (Example 28)
SEQ ID NO: 13: Second oligonucleotide + first oligonucleotide, 3'oligo (2'H-Uridine) + 5'oligo (cytidine) (Example 28)
SEQ ID NO: 14: Second oligonucleotide + first oligonucleotide, 3'oligo (2'H-Uridine) + 5'oligo (2'O-methyl-cytidine) (Example 28)
SEQ ID NO: 15: Second oligonucleotide, 3'oligo (2'H-Uridine) (Example 28)
Sequence number 16: RNA template (tmr27-II)
Sequence number 17: DNA template (tmd27-II)

Claims (67)

  1. 　第一のオリゴヌクレオチドまたはその改変体と第二のオリゴヌクレオチドまたはその改変体とを連結させる方法であって、
    　該第一のオリゴヌクレオチドまたはその改変体と該第二のオリゴヌクレオチドまたはその改変体とを、連結が生じる条件下に配置する工程を含み、
    　該第一のオリゴヌクレオチドまたはその改変体の３’末端と、該第二のオリゴヌクレオチドまたはその改変体の５’末端において連結が生じ、第二のオリゴヌクレオチドの５’末端側の末端モノマー部分が少なくとも一つの改変を含む、
    方法。     A method for linking a first oligonucleotide or a variant thereof and a second oligonucleotide or a variant thereof, the method comprising:
    arranging the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof under conditions in which linkage occurs;
    A linkage occurs between the 3' end of the first oligonucleotide or its variant and the 5' end of the second oligonucleotide or its variant, and the terminal monomer portion on the 5' end side of the second oligonucleotide is containing at least one modification;
    Method.
  2. 　前記第一のオリゴヌクレオチドまたはその改変体の３’末端は、リン酸基またはチオリン酸基を含む、請求項１に記載の方法。 The method according to claim 1, wherein the 3' end of the first oligonucleotide or its variant comprises a phosphate group or a thiophosphate group.
  3. 　前記第二のオリゴヌクレオチドまたはその改変体は、置換されていてもよいアミノ基を含む、請求項１に記載の方法。 The method according to claim 1, wherein the second oligonucleotide or a variant thereof contains an optionally substituted amino group.
  4. 　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の５’位に置換されていてもよいアミノ基を含む、請求項３に記載の方法。 4. The method according to claim 3, wherein the second oligonucleotide or its variant contains an optionally substituted amino group at the 5' position of its terminal monomer portion.
  5. 　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の５’位に置換されていないアミノ基を含む、請求項３および４に記載の方法。 5. The method according to claims 3 and 4, wherein the second oligonucleotide or variant thereof contains an unsubstituted amino group at the 5' position of its terminal monomer portion.
  6. 　前記アミノ基が、Ｃ１－６アルキル基によって置換されている、請求項３および４に記載の方法。 The method according to claims 3 and 4, wherein the amino group is substituted by a C 1-6 alkyl group.
  7. 　前記アミノ基がメチル基またはエチル基によって置換されている、請求項６に記載の方法。 7. The method according to claim 6, wherein the amino group is substituted with a methyl group or an ethyl group.
  8. 　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がハロまたは－ＯＨで置換されている、請求項１に記載の方法。 The method according to claim 1, wherein the second oligonucleotide or its variant has a terminal monomer portion substituted with halo or -OH at the 2' position.
  9. 　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がハロで置換されている、請求項８に記載の方法。 9. The method according to claim 8, wherein the second oligonucleotide or its variant has a halo substituted at the 2' position of its terminal monomer portion.
  10. 　前記第二のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がフルオロ基で置換されている、請求項９に記載の方法。 The method according to claim 9, wherein the second oligonucleotide or its variant has a fluoro group substituted at the 2' position of its terminal monomer portion.
  11. 　前記第一のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位が水素基または置換されていてもよいアルコキシ基で置換されている、請求項１に記載の方法。 The method according to claim 1, wherein the first oligonucleotide or its variant is substituted with a hydrogen group or an optionally substituted alkoxy group at the 2' position of its terminal monomer portion.
  12. 　前記第一のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位が置換されていてもよいアルコキシで置換されている、請求項１１に記載の方法。 The method according to claim 11, wherein the first oligonucleotide or its variant is substituted with an optionally substituted alkoxy at the 2' position of its terminal monomer portion.
  13. 　前記第一のオリゴヌクレオチドまたはその改変体は、その末端モノマー部分の２’位がメトキシで置換されている、請求項１２に記載の方法。 13. The method according to claim 12, wherein the first oligonucleotide or its variant is substituted with methoxy at the 2' position of its terminal monomer portion.
  14. 　前記末端モノマー部分の２’位が上向きで置換されている、請求項８から１３に記載の方法。 14. The method according to claims 8 to 13, wherein the 2' position of the terminal monomer moiety is upwardly substituted.
  15. 　前記末端モノマー部分の２’位が下向きで置換されている、請求項８から１３に記載の方法。 14. The method according to claims 8 to 13, wherein the 2' position of the terminal monomer moiety is substituted downward.
  16. 　前記第一のオリゴヌクレオチドまたはその改変体と前記第二のオリゴヌクレオチドまたはその改変体が、リン酸基またはチオリン酸基を介して連結している、請求項１に記載の方法。 The method according to claim 1, wherein the first oligonucleotide or its variant and the second oligonucleotide or its variant are linked via a phosphate group or a thiophosphate group.
  17. 　前記連結が生じる条件は、テンプレート核酸を用いることを包含する、請求項１に記載の方法。 The method according to claim 1, wherein the conditions under which the ligation occurs includes using a template nucleic acid.
  18. 　前記テンプレート核酸が、前記第一のオリゴヌクレオチドまたはその改変体および前記第二のオリゴヌクレオチドまたはその改変体と連続して相補的な配列および鎖長を有する、請求項１７に記載の方法。 The method according to claim 17, wherein the template nucleic acid has a sequence and chain length that are continuous and complementary to the first oligonucleotide or its variant and the second oligonucleotide or its variant.
  19. 　前記テンプレート核酸の鎖長が、１０残基以上である、請求項１８に記載の方法。 The method according to claim 18, wherein the template nucleic acid has a chain length of 10 residues or more.
  20. 　前記テンプレート核酸の鎖長が、２０～４０残基である、請求項１９に記載の方法。 The method according to claim 19, wherein the template nucleic acid has a chain length of 20 to 40 residues.
  21. 　前記テンプレート核酸が、前記第一のオリゴヌクレオチドもしくはその改変体および前記第二のオリゴヌクレオチドもしくはその改変体とは連結していないか、または、いずれか一方のオリゴヌクレオチドもしくはその改変体、もしくは両方のオリゴヌクレオチドもしくはその改変体と連結している、請求項１７～２０に記載の方法。 The template nucleic acid is not linked to the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof, or either one of the oligonucleotides or a variant thereof, or both. The method according to claims 17 to 20, wherein the oligonucleotide or a variant thereof is linked.
  22. 　前記テンプレート核酸の配列は、前記第一のオリゴヌクレオチドまたはその改変体および前記第二のオリゴヌクレオチドまたはその改変体と相補的な配列、あるいは二本鎖の形成を妨げない範囲のミスマッチ塩基対を含む、請求項２１に記載の方法。 The sequence of the template nucleic acid includes a sequence complementary to the first oligonucleotide or a variant thereof and the second oligonucleotide or a variant thereof, or mismatched base pairs within a range that does not prevent the formation of double strands. 22. The method of claim 21.
  23. 　前記連結が生じる条件は、オリゴヌクレオチド連結反応の活性化剤の使用を包含する、請求項１に記載の方法。 2. The method of claim 1, wherein the conditions under which the ligation occurs include the use of an activator for oligonucleotide ligation reactions.
  24. 　前記第一のオリゴヌクレオチドまたはその改変体が、下記式（Ｉ）

    （式中、Ｒ－はＳ－またはＯ－であり、Ｙは水素基またはアルコキシ基であり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｎは１以上の整数であり、Ｒ１は５’－位が水酸基である核酸、または核酸誘導体である）
    で示される化合物である、請求項１に記載の方法。     The first oligonucleotide or its variant has the following formula (I)

    (In the formula, R - is S - or O - , Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine) , 5-methylcytosine, N6-methyladenine, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-amino purine, or 2,6-diaminopurine, A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, n is an integer greater than or equal to 1, and R 1 is a hydroxyl group at the 5'-position. a certain nucleic acid or a nucleic acid derivative)
    The method according to claim 1, which is a compound represented by:
  25. 　前記Ｙがメトキシ基である、請求項２４に記載の方法。 The method according to claim 24, wherein the Y is a methoxy group.
  26. 　ｎが５から１０００の整数である、請求項２４に記載の方法。 25. The method according to claim 24, wherein n is an integer from 5 to 1000.
  27. 　前記第二のオリゴヌクレオチドまたはその改変体が、下記式（ＩＩ）

    （式中、Ｘは、ヒドロキシ基またはハロゲンであり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｐは１以上の整数であり、Ｒ２は３’－位が水酸基、リン酸基またはチオリン酸基である核酸、または核酸誘導体である）
    で示される化合物である、請求項１に記載の方法。     The second oligonucleotide or its variant has the following formula (II)

    (wherein, 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine , A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, p is an integer greater than or equal to 1, and R 2 is a nucleic acid in which the 3′-position is a hydroxyl group, a phosphate group, or a thiophosphate group, or a nucleic acid derivative)
    The method according to claim 1, which is a compound represented by:
  28. 　前記Ｘがフルオロ基である、請求項２７に記載の方法。 The method according to claim 27, wherein the X is a fluoro group.
  29. 　ｐが５から１０００の整数である、請求項２７に記載の方法。 28. The method of claim 27, wherein p is an integer from 5 to 1000.
  30. 　前記スペーサーが置換されていてもよいＣ１－１０アルキレンまたはＰＥＧである、請求項２４または２７に記載の方法。 28. The method of claim 24 or 27, wherein the spacer is an optionally substituted C 1-10 alkylene or PEG.
  31. 　前記スペーサーがＣ３アルキレンである、請求項３０に記載の方法。 31. The method of claim 30, wherein the spacer is a C3 alkylene.
  32. 　前記活性化剤が、カルボジイミド系縮合剤、ホスホニウム系縮合剤、ウロニウム系縮合剤またはトリアジン系縮合剤である、請求項２３に記載の方法。 The method according to claim 23, wherein the activator is a carbodiimide condensing agent, a phosphonium condensing agent, a uronium condensing agent, or a triazine condensing agent.
  33. 　前記カルボジイミド系縮合剤が１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）、ジイソプロピルカルボジイミド（ＤＩＰＣＩ）またはジシクロヘキシルカルボジイミド（ＤＣＣ）である、請求項３２に記載の方法。 The method according to claim 32, wherein the carbodiimide condensing agent is 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC), diisopropylcarbodiimide (DIPCI), or dicyclohexylcarbodiimide (DCC).
  34. 　前記ホスホニウム系縮合剤がブロモトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＢｒｏＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ピロリジン－１－イル）ホスホニウム・ヘキサフルオロホスファート（ＰｙＢＯＰ）、６－トリフルオロメチル－１Ｈ－ベンゾトリアゾール－１－イルオキシトリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＦＯＰ）、１Ｈ－ベンゾトリアゾール－１－イルオキシトリス（ジメチルアミノ）　ホスホニウムヘキサフルオロホスファート（ＢＯＰ）または（７－アザベンゾトリアゾール－１－イルオキシ）トリピロリジノホスホニウムヘキサフルオロホスファート（ＰｙＡＯＰ）である、請求項３２に記載の方法。 The phosphonium condensing agent is bromotripyrrolidinophosphonium hexafluorophosphate (PyBroP), 1H-benzotriazol-1-yloxytris(pyrrolidin-1-yl)phosphonium hexafluorophosphate (PyBOP), 6-trifluoro Methyl-1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyFOP), 1H-benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) or (7-aza 33. The method of claim 32, wherein the method is benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyAOP).
  35. 　前記ウロニウム系縮合剤がＯ－（７－アザベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（ＨＡＴＵ）、Ｏ－（ベンゾトリアゾール－１－イル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウムヘキサフルオロホスファート（ＨＢＴＵ）、Ｏ－（Ｎ－スクシンイミジル）－Ｎ，Ｎ，Ｎ’，Ｎ’－テトラメチルウロニウム　テトラフルオロほう酸塩（ＴＳＴＵ）または｛｛［（１－シアノ－２－エトキシ－２－オキソエチリデン）アミノ］オキシ｝－４－モルホリノメチレン｝ジメチルアンモニウム　ヘキサフルオロホスファート（ＣＯＭＵ）である、請求項３２に記載の方法。 The uronium-based condensing agent is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(benzotriazole-1- )-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroboric acid salt (TSTU) or {{[(1-cyano-2-ethoxy-2-oxoethylidene)amino]oxy}-4-morpholinomethylene}dimethylammonium hexafluorophosphate (COMU) according to claim 32. Method.
  36. 　前記トリアジン系縮合剤がＤＭＴ－ＭＭである、請求項３２に記載の方法。 The method according to claim 32, wherein the triazine condensing agent is DMT-MM.
  37. 　前記活性化剤が、１－フルオロ－２，４－ジニトロベンゼン（ＤＮＦＢ）、または１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）である、請求項２１に記載の方法。 22. The method of claim 21, wherein the activator is 1-fluoro-2,4-dinitrobenzene (DNFB) or 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC). .
  38. 　前記活性化剤が、１－［３－（ジメチルアミノ）プロピル］－３－エチルカルボジイミド（ＥＤＣ）と１－ヒドロキシ－７－アザベンゾトリアゾール（ＨＯＡｔ）、１－ヒドロキシベンゾトリアゾール（ＨＯＢｔ）またはＮ－ヒドロキシコハク酸イミド（ＨＯＳｕ）とを組み合わせて使用される、請求項３２～３７に記載の方法。 The activator may be 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and 1-hydroxy-7-azabenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt) or N- The method according to claims 32 to 37, used in combination with hydroxysuccinimide (HOSu).
  39. 　前記第一のオリゴヌクレオチドまたはその改変体の５’末端と前記第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体の複数と、前記第一のオリゴヌクレオチドまたはその改変体と、前記第二のオリゴヌクレオチドまたはその改変体とが、2か所以上同時にまたは段階的に連結される、請求項１に記載の方法。 a plurality of oligonucleotides or variants thereof in which the 5' end of the first oligonucleotide or variant thereof and the 3' end of the second oligonucleotide or variant thereof are linked, and the first oligonucleotide or variant thereof; 2. The method according to claim 1, wherein the variant and the second oligonucleotide or the variant thereof are linked at two or more locations simultaneously or in a stepwise manner.
  40. 　前記第一のオリゴヌクレオチドまたはその改変体の５’末端および前記テンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、前記第二のオリゴヌクレオチドまたはその改変体と連結される、請求項１に記載の方法。 The oligonucleotide or variant thereof to which the 5' end of the first oligonucleotide or variant thereof and the template nucleic acid are linked is linked to the second oligonucleotide or variant thereof. Method.
  41. 　前記第二のオリゴヌクレオチドまたはその改変体の３’末端および前記テンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、前記第一のオリゴヌクレオチドまたはその改変体と連結される、請求項１に記載の方法。 The oligonucleotide or variant thereof to which the 3' end of the second oligonucleotide or variant thereof and the template nucleic acid are linked is linked to the first oligonucleotide or variant thereof. Method.
  42. 　前記第一のオリゴヌクレオチドまたはその改変体の５’末端と第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体と、前記第一のオリゴヌクレオチドまたはその改変体の３’末端と、第二のオリゴヌクレオチドまたはその改変体の５’末端とが、連結される、請求項１に記載の方法。 An oligonucleotide or a variant thereof in which the 5' end of the first oligonucleotide or a variant thereof is linked to the 3' end of a second oligonucleotide or a variant thereof, and the first oligonucleotide or a variant thereof. The method according to claim 1, wherein the 3' end of the second oligonucleotide or the 5' end of the second oligonucleotide or a variant thereof are linked.
  43. 　Ｒ２の３’－位がリン酸基またはチオリン酸基である第二のオリゴヌクレオチドまたはその改変体の複数が、複数のテンプレート核酸をもちいて、環状に連結される、請求項２７に記載の方法。 The method according to claim 27, wherein a plurality of second oligonucleotides or variants thereof in which the 3'-position of R2 is a phosphate group or a thiophosphate group are linked in a circular manner using a plurality of template nucleic acids. .
  44. 　前記テンプレート核酸が前記第一のオリゴヌクレオチドまたはその改変体の５’末端および前記第二のオリゴヌクレオチドまたはその改変体の３’末端と連結したオリゴヌクレオチドまたはその改変体において、該第一のオリゴヌクレオチドまたはその改変体の５’末端と該第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結される、請求項１に記載の方法。 In the oligonucleotide or a variant thereof, in which the template nucleic acid is linked to the 5' end of the first oligonucleotide or the variant thereof and the 3' end of the second oligonucleotide or the variant thereof, the first oligonucleotide The method according to claim 1, wherein the 5' end of the second oligonucleotide or the variant thereof is linked to the 3' end of the second oligonucleotide or the variant thereof.
  45. 　請求項１に記載の第一のオリゴヌクレオチドまたはその改変体または請求項１に記載の第二のオリゴヌクレオチドまたはその改変体を含む、それぞれ該第二のオリゴヌクレオチドまたはその改変体または該第一のオリゴヌクレオチドまたはその改変体を用いて連結オリゴヌクレオチドまたはその改変体を製造するための組成物。 The first oligonucleotide or a variant thereof according to claim 1 or the second oligonucleotide or a variant thereof according to claim 1, respectively. A composition for producing a linked oligonucleotide or a variant thereof using an oligonucleotide or a variant thereof.
  46. 　請求項１に記載の第一のオリゴヌクレオチドまたはその改変体もしくは請求項１に記載の第二のオリゴヌクレオチドまたはその改変体、またはそれらの組み合わせ
    を含む、テンプレート核酸を用いて連結オリゴヌクレオチドを製造するための組成物。     Producing a linked oligonucleotide using a template nucleic acid comprising the first oligonucleotide according to claim 1 or a variant thereof, the second oligonucleotide according to claim 1 or a variant thereof, or a combination thereof. Composition for.
  47. 　オリゴヌクレオチドまたはその改変体を連結するためのキットであって、
    請求項１に記載の第一のオリゴヌクレオチドまたはその改変体と、
    請求項１に記載の第二のオリゴヌクレオチドまたはその改変体と、
    活性化剤と、
    テンプレート核酸と
    を含むキット。     A kit for linking oligonucleotides or variants thereof, comprising:
    The first oligonucleotide according to claim 1 or a variant thereof;
    the second oligonucleotide according to claim 1 or a variant thereof;
    an activator;
    A kit comprising a template nucleic acid.
  48. 　請求項１に記載の第一のオリゴヌクレオチドまたはその改変体および
    請求項１に記載の第二のオリゴヌクレオチドまたはその改変体
    を含む、連結オリゴヌクレオチドを製造するための原料組成物。     A raw material composition for producing a linked oligonucleotide, comprising the first oligonucleotide according to claim 1 or a variant thereof and the second oligonucleotide according to claim 1 or a variant thereof.
  49. 　前記第二のオリゴヌクレオチドまたはその改変体を製造するための原料が、２’位にフルオロ基、５’位にアミノ基を有するヌクレオシドを含む、請求項４８に記載の原料組成物。 The raw material composition according to claim 48, wherein the raw material for producing the second oligonucleotide or a variant thereof contains a nucleoside having a fluoro group at the 2' position and an amino group at the 5' position.
  50. 　第一および／または第二のオリゴヌクレオチドまたはその改変体の原料（モノマーまたは置換基導入試薬等）を含む、
    請求項１に記載の第一のオリゴヌクレオチドもしくはその改変体、および／または
    請求項１に記載の第二のオリゴヌクレオチドもしくはその改変体を製造するための組成物。     Containing raw materials (monomers or substituent introduction reagents, etc.) for the first and/or second oligonucleotides or variants thereof;
    A composition for producing the first oligonucleotide according to claim 1 or a variant thereof, and/or the second oligonucleotide according to claim 1 or a variant thereof.
  51. 　前記第一および／または第二のオリゴヌクレオチドまたはその改変体の原料が、アミダイド体のまたはデオキシヌクレオシド、または３’位が水酸基であるまたはデオキシヌクレオシドである、請求項５０に記載の組成物。 The composition according to claim 50, wherein the raw material for the first and/or second oligonucleotide or a variant thereof is an amidide or deoxynucleoside, or a deoxynucleoside having a hydroxyl group at the 3' position.
  52. Ａ）合成を所望する核酸配列の情報を得る工程、
    Ｂ）Ａ）の情報に基づき、合成したい配列の一部を複数合成する工程であって、ここで、各配列は、請求項１に記載の特徴を有する、工程、ならびに
    Ｃ）必要に応じて連結反応に必要な試薬の存在下で、連結条件に配置する工程
    を包含する、所望の核酸配列を有するオリゴヌクレオチドまたはその改変体の製造方法。     A) obtaining information on the nucleic acid sequence desired to be synthesized;
    B) A step of synthesizing a plurality of parts of the sequence to be synthesized based on the information in A), where each sequence has the characteristics according to claim 1, and C) as necessary. A method for producing an oligonucleotide having a desired nucleic acid sequence or a variant thereof, which comprises the step of placing the oligonucleotide under ligation conditions in the presence of a reagent necessary for the ligation reaction.
  53. Ａ）合成を所望する核酸配列の情報を得る工程、
    Ｂ）Ａ）の情報に基づき、合成したい配列の一部を複数合成する工程であって、ここで、各配列は、請求項１に記載の特徴を有する、工程、ならびに
    Ｃ）必要に応じて連結反応に必要な試薬と、Ｂ）で合成された部分配列を含む核酸とをパッケージとする工程
    を包含する、所望のオリゴヌクレオチドまたはその改変体の製造キットの製造方法。     A) obtaining information on the nucleic acid sequence desired to be synthesized;
    B) A step of synthesizing a plurality of parts of the sequence to be synthesized based on the information in A), where each sequence has the characteristics according to claim 1, and C) as necessary. A method for producing a kit for producing a desired oligonucleotide or a variant thereof, which includes the step of packaging the reagents necessary for the ligation reaction and the nucleic acid containing the partial sequence synthesized in B).
  54. 　以下の式

    （式中、Ｙは、水素基またはアルコキシ基であり、Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、Ｘは、ヒドロキシ基、またはハロゲンであり、Ａは、各出現において独立してスペーサー、核酸、または核酸誘導体であり、ｎは１以上の整数であり、ｐは１以上の整数であり、Ｒ１は５’－位が水酸基である核酸、もしくは核酸誘導体、またはＲ１は２’－位が水素基またはアルコキシ基であり、５’－位がアミノ基である核酸、もしくは核酸誘導体であり、Ｒ２は３’－位が水酸基、リン酸基もしくはチオリン酸基である核酸、もしくは核酸誘導体、またはＲ２は２’－位がヒドロキシ基、またはハロゲンであり、３’－位がリン酸基もしくはチオリン酸基である核酸、もしくは核酸誘導体であるか、Ｒ１およびＲ２は、一緒になって単結合を形成してもよく、Ｒ１およびＲ２は、異なる化合物のＲ１およびＲ２と結合してもよく、Ｒ３が置換されていてもよいアミノ基である）で示される化合物。     The following formula

    (In the formula, Y is a hydrogen group or an alkoxy group, and B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine , 1-methyladenine, pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine. , X is a hydroxy group or a halogen, A is independently at each occurrence a spacer, a nucleic acid, or a nucleic acid derivative, n is an integer of 1 or more, p is an integer of 1 or more, R 1 is a nucleic acid or a nucleic acid derivative in which the 5'-position is a hydroxyl group, or R 1 is a nucleic acid or a nucleic acid derivative in which the 2'-position is a hydrogen group or an alkoxy group and the 5'-position is an amino group. , R 2 is a nucleic acid or a nucleic acid derivative whose 3'-position is a hydroxyl group, phosphate group, or thiophosphoric acid group, or R 2 is a hydroxyl group or a halogen at the 2'-position and a phosphoric acid group at the 3'-position. R 1 and R 2 may be taken together to form a single bond, and R 1 and R 2 are R 1 and R 2 of different compounds. 2 and R 3 is an optionally substituted amino group.
  55. 　前記Ｙは、メトキシ基であり、前記Ｂは、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、ウラシル、ヒポキサンチン（イノシン）、５－メチルシトシン、Ｎ６－メチルアデニン、１－メチルアデニン、シュードウラシル、１－メチルシュードウラシル、５－ブロモウラシル、５－ヨードウラシル、６－チオアデニン、６－チオグアニン、４－チオウラシル、２－アミノプリン、または２，６－ジアミノプリンであり、前記Ｘは、フルオロ基であり、Ｒ１およびＲ２は、一緒になって単結合を形成している、請求項５４に記載の化合物。 Said Y is a methoxy group, and said B is each independently adenine, cytosine, methylcytosine, guanine, thymine, uracil, hypoxanthine (inosine), 5-methylcytosine, N6-methyladenine, 1-methyladenine. , pseudouracil, 1-methylpseudouracil, 5-bromouracil, 5-iodouracil, 6-thioadenine, 6-thioguanine, 4-thiouracil, 2-aminopurine, or 2,6-diaminopurine, and the X is , a fluoro group, and R 1 and R 2 are taken together to form a single bond.
  56. 　ｎが５から１０００の整数であり、ｐが５から１０００の整数である、請求項５４に記載の化合物。 55. The compound according to claim 54, wherein n is an integer from 5 to 1000 and p is an integer from 5 to 1000.
  57. 　前記Ｒ３が、置換されていないアミノ基である、請求項５４に記載の化合物。 55. The compound according to claim 54, wherein R3 is an unsubstituted amino group.
  58. 　前記Ｒ３が、Ｃ１－６アルキル基によって置換されたアミノ基である、請求項５４に記載の化合物。 55. The compound according to claim 54, wherein R 3 is an amino group substituted with a C 1-6 alkyl group.
  59. 　前記Ｒ３がメチル基またはエチル基によって置換されたアミノ基である、請求項５８に記載の化合物。 59. The compound according to claim 58, wherein R3 is an amino group substituted with a methyl group or an ethyl group.
  60. 　前記Ｂが、それぞれ独立してアデニン、シトシン、メチルシトシン、グアニン、チミン、またはウラシルである、請求項５４に記載の化合物。 55. The compound of claim 54, wherein B is each independently adenine, cytosine, methylcytosine, guanine, thymine, or uracil.
  61. 　前記Ｒ１および前記Ｒ２が、一緒になって単結合を形成している、請求項５４に記載の化合物。 55. The compound of claim 54, wherein R1 and R2 are taken together to form a single bond.
  62. 　請求項１に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端と請求項１に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体の複数と、前記第一のオリゴヌクレオチドまたはその改変体と、前記第二のオリゴヌクレオチドまたはその改変体とが、連結されている、連結オリゴヌクレオチドまたはその改変体。 An oligonucleotide or a variant thereof in which the 5' end of the first oligonucleotide according to claim 1 or a variant thereof is linked to the 3' end of the second oligonucleotide according to claim 1 or a variant thereof. A linked oligonucleotide or a variant thereof, in which a plurality of oligonucleotides, the first oligonucleotide or a variant thereof, and the second oligonucleotide or a variant thereof are linked.
  63. 　請求項１に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端および請求項１に記載のテンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、請求項１に記載の第二のオリゴヌクレオチドまたはその改変体と連結されている、連結オリゴヌクレオチドまたはその改変体。 The 5' end of the first oligonucleotide according to claim 1 or a variant thereof and the template nucleic acid according to claim 1 linked to the oligonucleotide or a variant thereof is the second oligonucleotide according to claim 1. or a variant thereof, a linked oligonucleotide or a variant thereof.
  64. 　請求項１に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端および請求項１に記載のテンプレート核酸が連結したオリゴヌクレオチドまたはその改変体が、請求項１に記載のオリゴヌクレオチドまたはその改変体と連結されている、連結オリゴヌクレオチドまたはその改変体。 The oligonucleotide or the variant thereof in which the 3' end of the second oligonucleotide according to claim 1 or the variant thereof and the template nucleic acid according to claim 1 are linked is the oligonucleotide according to claim 1 or the variant thereof. A linking oligonucleotide or a variant thereof, which is linked to a body.
  65. 　請求項１に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端と請求項１に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結したオリゴヌクレオチドまたはその改変体と、前記第一のオリゴヌクレオチドまたはその改変体の３’末端と、第二のオリゴヌクレオチドまたはその改変体の５’末端とが、連結されている、連結オリゴヌクレオチドまたはその改変体。 An oligonucleotide or a variant thereof in which the 5' end of the first oligonucleotide according to claim 1 or a variant thereof is linked to the 3' end of the second oligonucleotide according to claim 1 or a variant thereof. , a linked oligonucleotide or a variant thereof, wherein the 3' end of the first oligonucleotide or the variant thereof and the 5' end of the second oligonucleotide or the variant thereof are linked.
  66. 　請求項１に記載のテンプレート核酸が請求項１に記載の第一のオリゴヌクレオチドまたはその改変体の５’末端および請求項１に記載の第二のオリゴヌクレオチドまたはその改変体の３’末端と連結したオリゴヌクレオチドまたはその改変体において、該第一のオリゴヌクレオチドまたはその改変体の５’末端と該第二のオリゴヌクレオチドまたはその改変体の３’末端とが連結されている、連結オリゴヌクレオチドまたはその改変体。 The template nucleic acid according to claim 1 is linked to the 5' end of the first oligonucleotide according to claim 1 or a variant thereof and the 3' end of the second oligonucleotide according to claim 1 or a variant thereof A linked oligonucleotide or its variant, in which the 5' end of the first oligonucleotide or its variant and the 3' end of the second oligonucleotide or its variant are linked. variant.
  67. 　請求項５４から６１のいずれか一項に記載の化合物を含む、医薬組成物。 A pharmaceutical composition comprising a compound according to any one of claims 54 to 61.
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