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Method of suppressing formation of photocrosslink, and photoreactive nucleic acid in which auto-crosslink formation is suppressed

外国特許コード F170009046
整理番号 J1019-03WO
掲載日 2017年4月26日
出願国 アメリカ合衆国
出願番号 201415033422
公報番号 20160326207
出願日 平成26年10月30日(2014.10.30)
公報発行日 平成28年11月10日(2016.11.10)
国際出願番号 JP2014078980
国際公開番号 WO2015064718
国際出願日 平成26年10月30日(2014.10.30)
国際公開日 平成27年5月7日(2015.5.7)
優先権データ
  • 特願2013-225799 (2013.10.30) JP
  • 2014WO-JP78980 (2014.10.30) WO
発明の名称 (英語) Method of suppressing formation of photocrosslink, and photoreactive nucleic acid in which auto-crosslink formation is suppressed
発明の概要(英語) (US20160326207)
Provided is a means for preventing the inactivation of a photoresponsive nucleic acid probe by suppressing the formation of a photocrosslink between a photoresponsive base having a photocrosslinkable vinyl structure and a photocrosslinkable thymine (T) or uracil (U) base, by substituting with an R group (R being —CN or —CO—R1, where R1 is a saturated or unsaturated straight-chain or branched cyclic or non-cyclic C1-12 hydrocarbon group) the 5 position of a pyrimidine ring of the thymine (T) or uracil (U) base which is photocrosslinkable with the photoresponsive base having a photocrosslinkable vinyl structure.
特許請求の範囲(英語) [claim1]
1. A method of suppressing photo-crosslinkage of a photoresponsive base having a photo-crosslinkable vinyl structure with a thymine (T) or uracil (U) base that is photo-crosslinkable to the photoresponsive base having a photo-crosslinkable vinyl structure, comprising: substituting C5 of a pyrimidine ring of the thymine (T) or uracil (U) base with an R group (R is -- CN or -- CO -- R1, and R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group).
[claim2]
2. The method according to claim 1, wherein the photo-crosslinkage is a reaction in which a photo-crosslink is formed between:
the photoresponsive base having a photo-crosslinkable vinyl structure being contained in a base sequence of a photoresponsive nucleic acid, and
the thymine (T) or uracil (U) base that is photo-crosslinkable to the photoresponsive base, the thymine (T) or uracil (U) base being contained in a base sequence fraction complementary to a base sequence fraction that is contained in the base sequence of the photoresponsive nucleic acid and is composed of 4 or more bases including the photoresponsive base (the base sequence fraction that is contained in the base sequence of the photoresponsive nucleic acid and is composed of 4 or more bases including the photoresponsive base is called a photoresponsive base sequence fraction) (the base sequence fraction complementary to the photoresponsive base sequence fraction is called a complementary base sequence fraction), and the complementary base sequence fraction being contained in a nucleic acid (a partially-complementary nucleic acid), and
in the complementary base sequence fraction contained in the partially-complementary nucleic acid, at least one constituent nucleotide containing the photo-crosslinkable T or U is replaced by a modified nucleotide of Formula (I):
(in Formula I,
R is -- CN or -- CO -- R1,
R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group,
X forms a phosphate group together with O that is bonded to X in Formula I,
Y is a hydroxy group, and
Z is hydrogen or a hydroxy group), the modified nucleotide of Formula (I) being introduced to the complementary base sequence fraction by a phosphodiester bond, and as a result, photo-crosslinkage of the photoresponsive nucleic acid with the partially-complementary nucleic acid is suppressed.
[claim3]
3. The method according to claim 1, wherein the photoresponsive nucleic acid contains both the photoresponsive base sequence fraction and the complementary base sequence fraction as separate sequence regions within a molecule of the photoresponsive nucleic acid, the molecule of the photoresponsive nucleic acid being the same as a molecule of the partially-complementary nucleic acid, and
suppression of the photo-crosslinkage of the photoresponsive nucleic acid with the partially-complementary nucleic acid is achieved by suppressing self-crosslinkage within the photoresponsive nucleic acid caused by the photo-crosslinkage of the photoresponsive base with the photo-crosslinkable thymine (T) or uracil (U) base.
[claim4]
4. The method according to claim 1, wherein R in Formula (I) is -- CN.
[claim5]
5. The method according to claim 1, wherein the photoresponsive base having a photo-crosslinkable vinyl structure is a photoresponsive base having a 3-vinylcarbazole structure.
[claim6]
6. The method according to claim 1, wherein the photoresponsive base having a photo-crosslinkable vinyl structure is a base portion of a modified nucleotide of Formula (II) or Formula (III):
(in Formula II, Ra is a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or hydrogen,
R2 and R3 are independently a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or hydrogen,
X forms a phosphate group together with O that is bonded to X in Formula II,
Y is a hydroxy group, and
Z is hydrogen or a hydroxy group);
(in Formula II,
Ra is a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, a phosphono group, a sulfo group, or a hydrogen atom,
R2 and R3 are independently a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R4 is a hydrogen atom, a hydroxy group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a fluoromethyl group, a monovalent group of a C6-C12 monocyclic or dicyclic aromatic compound, a monovalent group of a monocyclic or dicyclic, C6-C12 heterocyclic aromatic compound, or a monovalent group of a formula:
(wherein Ra, R2, and R3 are independent of Ra, R2, and R3 as defined for Formula III and are selected from the groups given above as examples of Ra, R2, and R3 for Formula II),
R6 is a hydrogen atom, a methyl group, or an ethyl group,
Q1 forms a phosphate group together with O that is bonded to Q1 in Formula III, and
Q2 is a hydrogen atom), and
the photoresponsive base having a photo-crosslinkable vinyl structure is introduced into the photoresponsive base sequence fraction as a base portion of the modified nucleotide by a phosphodiester bond of the modified nucleotide.
[claim7]
7. The method according to claim 1, wherein every nucleotide containing T or U in the base sequence of the partially-complementary nucleic acid is substituted with the modified nucleotide of Formula (I).
[claim8]
8. A photoresponsive nucleic acid with suppressed self-crosslinking ability, comprising: a photoresponsive base having a photo-crosslinkable vinyl structure, and
a modified nucleotide of Formula (I):
(in Formula I,
R is -- CN or -- CO -- R1,
R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 hydrocarbon group,
X forms a phosphate group together with O that is bonded to X in Formula I,
Y is a hydroxy group, and
Z is hydrogen or a hydroxy group), in place of at least one constituent nucleotide containing photo-crosslinkable T or U.
[claim9]
9. The photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 8, wherein R in Formula (I) is -- CN.
[claim10]
10. The photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 8, wherein the photoresponsive base having a photo-crosslinkable vinyl structure is a photoresponsive base having a 3-vinylcarbazole structure.
[claim11]
11. The photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 8, wherein the photoresponsive base having a photo-crosslinkable vinyl structure is a base portion of a modified nucleotide of Formula (II) or Formula (III):
(in Formula II, Ra is a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or hydrogen,
R2 and R3 are independently a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or hydrogen,
X forms a phosphate group together with O that is bonded to X in Formula II,
Y is a hydroxy group, and
Z is hydrogen or a hydroxy group);
(in Formula II,
Ra is a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, a phosphono group, a sulfo group, or a hydrogen atom,
R2 and R3 are independently a cyano group, an amido group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R4 is a hydrogen atom, a hydroxy group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a fluoromethyl group, a monovalent group of a C6-C12 monocyclic or dicyclic aromatic compound, a monovalent group of a monocyclic or dicyclic, C6-C12 heterocyclic aromatic compound, or a monovalent group of a formula:
(wherein Ra, R2, and R3 are independent of Ra, R2, and R3 as defined for Formula III and are selected from the groups given above as examples of Ra, R2, and R3 for Formula III),
R6 is a hydrogen atom, a methyl group, or an ethyl group,
Q1 forms a phosphate group together with O that is bonded to Q1 in Formula III, and
Q2 is a hydrogen atom), and
the photoresponsive base having a photo-crosslinkable vinyl structure is introduced into the photoresponsive base sequence fraction as a base portion of the modified nucleotide by a phosphodiester bond of the modified nucleotide.
[claim12]
12. The photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 8, wherein the photoresponsive nucleic acid with suppressed self-crosslinking ability comprises a base sequence fraction complementary to a base sequence fraction composed of 4 or more bases including the photoresponsive base (the base sequence fraction composed of 4 or more bases including the photoresponsive base is called a photoresponsive base sequence fraction) (the base sequence fraction complementary to the photoresponsive base sequence fraction is called a complementary base sequence fraction), and
the photo-crosslinkable T or U is T or U contained in the complementary base sequence fraction.
[claim13]
13. The photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 8, wherein every nucleotide containing T or U in the base sequence of the photoresponsive nucleic acid with suppressed self-crosslinking ability is substituted with the modified nucleotide of Formula (I).
  • 発明者/出願人(英語)
  • FUJIMOTO KENZO
  • NAKAMURA SHIGETAKA
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
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