TOP > 外国特許検索 > Method of suppressing formation of photocrosslink, and photoreactive nucleic acid in which auto-crosslink formation is suppressed

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

Foreign code F170009046
File No. J1019-03WO
Posted date 2017年4月26日
Country アメリカ合衆国
Application number 201415033422
Gazette No. 20160326207
Gazette No. 10844087
Date of filing 平成26年10月30日(2014.10.30)
Gazette Date 平成28年11月10日(2016.11.10)
Gazette Date 令和2年11月24日(2020.11.24)
International application number JP2014078980
International publication number WO2015064718
Date of international filing 平成26年10月30日(2014.10.30)
Date of international publication 平成27年5月7日(2015.5.7)
Priority data
  • 特願2013-225799 (2013.10.30) JP
  • 2014JP78980 (2014.10.30) WO
Title Method of suppressing formation of photocrosslink, and photoreactive nucleic acid in which auto-crosslink formation is suppressed
Abstract Provided is a means for preventing the inactivation of a photoresponsive nucleic acid probe by suppressing the formation of a photocrosslink between a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III) and a modified nucleotide having a structure corresponding to the monomer of Formula (I), wherein the modified nucleotide replaces at least one constituent nucleotide which is the photocrosslinkable 1-thyminyl or 1-uracilyl, by substituting at least one constituent nucleotide which is the photocrosslinkable 1-thyminyl or 1-uracilyl with a modified nucleotide having a structure corresponding to the monomer of Formula (I).
Outline of related art and contending technology BACKGROUND
As a basic technique in the field of molecular biology, formation and detection of a double strand of nucleic acids are widely used not only in basic research but also in the fields of medical care, industry, agriculture, and the like. A particularly useful technique used in formation and detection of a double-stranded nucleic acid is the photo-crosslinking technique with the use of photoresponsive nucleic acids. The photo-crosslinking technique is used in a wide range of applications, for example, in the field of medical care including antisense drugs and other nucleic acid drugs as well as SNP sensing, and the field of DNA nanotechnology with the use of nucleic acids. The photo-crosslinking technique with the use of photoresponsive nucleic acids has been developed by the inventors of the present invention and their research group, and a plurality of artificial photoresponsive nucleotides have been developed and are under patent pending (Patent Document 1).
One of the most remarkable applications of the photo-crosslinking technique is highly-sensitive selective amplification of a nucleic acid (the photo-clamping method) (Patent Document 2). This is a method of, prior to PCR amplification of a nucleic acid, using a photoresponsive nucleic acid as a clamp probe and subjecting the clamp probe to photo-linkage with a nucleic acid that has a wild-type (or normal) base sequence and is present in a large number in a specimen, so as to form an indissociable double-stranded nucleic acid, and as a result, suppressing PCR amplification of the large number of nucleic acid having a wild-type (or normal) base sequence, thereby allowing selective and highly sensitive amplification of a nucleic acid that has a target mutant base sequence and is present in a small amount in the specimen.
Scope of claims [claim1]
1. A double stranded nucleic acid, comprising:
a first oligonucleotide, the first oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III):
wherein, in Formula II,
Ra is a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R2 and R3 are independently a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
X forms a phosphate group together with O that is bonded to X,
Y is a hydroxy group, and
Z is a hydrogen atom or a hydroxyl group;
wherein, in Formula III,
Ra is a cyano 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, 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 group, a naphthyl group, an indolyl group, a benzoimidazolyl group, a benzothiophenyl group, 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, and
Q2 is a hydrogen atom, and
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) is introduced into a base sequence fraction as a base portion of the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) by a phosphodiester bond, and
a second oligonucleotide, the second oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
wherein, 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,
Y is a hydroxy group, and
Z is a hydrogen atom or a hydroxy group, and
wherein the first oligonucleotide and the second oligonucleotide are hybridized.

[claim2]
2. The double stranded nucleic acid according to claim 1, wherein R in Formula (I) is a cyano group.

[claim3]
3. The double stranded nucleic acid according to claim 2,
wherein the first oligonucleotide comprises 4 or more bases.

[claim4]
4. The double stranded nucleic acid according to claim 1,
wherein the first oligonucleotide comprises 4 or more bases.

[claim5]
5. The double stranded nucleic acid according to claim 4, wherein the modified nucleotide having the structure corresponding to the monomer of Formula (I) in the second oligonucleotide is at every position complementary to the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) in the first oligonucleotide.

[claim6]
6. The double stranded nucleic acid according to claim 1,
wherein the first oligonucleotide contains the modified nucleotide having the structure corresponding to the monomer of Formula (I), and
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (I) replaces at least one constituent nucleotide which is the photo-crosslinkable thymine (T) or uracil (U).

[claim7]
7. The double stranded nucleic acid according to claim 6, wherein R in Formula (I) is a cyano group.

[claim8]
8. The double stranded nucleic acid according to claim 7,
wherein the first oligonucleotide comprises 4 or more bases.

[claim9]
9. The double stranded nucleic acid according to claim 6, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.

[claim10]
10. The double stranded nucleic acid according to claim 9,
wherein the first oligonucleotide comprises 4 or more bases.

[claim11]
11. The double stranded nucleic acid according to claim 6,
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.

[claim12]
12. The double stranded nucleic acid according to claim 11,
wherein the first oligonucleotide comprises 4 or more bases.

[claim13]
13. The double stranded nucleic acid according to claim 6,
wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom, and
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.

[claim14]
14. The double stranded nucleic acid according to claim 13,
wherein the first oligonucleotide comprises 4 or more bases.

[claim15]
15. The double stranded nucleic acid according to claim 1, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.

[claim16]
16. The double stranded nucleic acid according to claim 15,
wherein the first oligonucleotide comprises 4 or more bases.

[claim17]
17. The double stranded nucleic acid according to claim 1,
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.

[claim18]
18. The double stranded nucleic acid according to claim 17,
wherein the first oligonucleotide comprises 4 or more bases.

[claim19]
19. The double stranded nucleic acid according to claim 1,
wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom, and
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.

[claim20]
20. The double stranded nucleic acid according to claim 19,
wherein the first oligonucleotide comprises 4 or more bases.

[claim21]
21. A single stranded oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
wherein, in Formula I:
R is -CO-R1,
R1 is a saturated or unsaturated, linear or branched, cyclic or acyclic, C1-C12 unsubstituted hydrocarbon group,
X forms a phosphate group together with O that is bonded to X,
Y is a hydroxy group, and
Z is a hydrogen atom or a hydroxy group.

[claim22]
22. The single stranded oligonucleotide according to claim 21, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.

[claim23]
23. A single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability comprising:
a first base sequence, the first base sequence comprising a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III):
wherein, in Formula II,
Ra is a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R2 and R3 are independently a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
X forms a phosphate group together with O that is bonded to X,
Y is a hydroxy group, and
Z is a hydrogen atom or a hydroxyl group;
wherein, in Formula III,
Ra is a cyano 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, 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 group, a monovalent group of a monocyclic or dicyclic, naphthyl group, indolyl group, benzoimidazolyl group, benzothiophenyl group 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, and
Q2 is a hydrogen atom, and
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) is introduced into a base sequence fraction as a base portion of the modified nucleotide of having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) by a phosphodiester bond,
and
a second base sequence, the second base sequence comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
wherein, 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,
Y is a hydroxy group, and
Z is a hydrogen atom or a hydroxy group.

[claim24]
24. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 23,
wherein R in Formula (I) is a cyano group.

[claim25]
25. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 24,
wherein R is a cyano group, and Z is a hydrogen atom.

[claim26]
26. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 25,
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.

[claim27]
27. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 25,
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).

[claim28]
28. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 24,
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.

[claim29]
29. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 28,
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.

[claim30]
30. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 28,
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).

[claim31]
31. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 24,
wherein R is a cyano group, and Z is a hydrogen atom,
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.

[claim32]
32. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 31,
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.

[claim33]
33. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 31,
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).

[claim34]
34. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 24,
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.

[claim35]
35. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 24,
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).

[claim36]
36. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 23,
wherein R is a cyano group, and Z is a hydrogen atom.

[claim37]
37. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 36,
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.

[claim38]
38. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 36,
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).

[claim39]
39. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 23,
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) wherein, in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2 is a hydrogen atom.

[claim40]
40. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 39,
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.

[claim41]
41. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 39,
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).

[claim42]
42. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 23,
wherein each of the first base sequence and the second base sequence comprises 4 or more bases.

[claim43]
43. The single stranded photoresponsive nucleic acid with suppressed self-crosslinking ability according to claim 23,
wherein every nucleotide containing T or U in the sequence of the single stranded photoresponsive nucleic acid with suppressed self-crosslinkable ability is substituted with the modified nucleotide having the structure corresponding to the monomer of Formula (I).

[claim44]
44. A method of hybridizing nucleic acid sequences, comprising:
performing a hybridizing reaction of a second oligonucleotide with a first oligonucleotide by mixing a first base sequence fraction including the first oligonucleotide with a second base sequence fraction including the second oligonucleotide, the second oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (I):
wherein, 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,
Y is a hydroxy group, and
Z is a hydrogen atom or a hydroxy group,
wherein the modified nucleotide having a structure corresponding to the monomer of Formula (I) replaces at least one constituent nucleotide which is the photo-crosslinkable thymine (T) or uracil (U),
the first oligonucleotide comprising a modified nucleotide having a structure corresponding to the monomer of Formula (II) or an amino acid analogue of a nucleotide having a structure corresponding to the monomer of Formula (III):
wherein, in Formula II,
Ra is a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R2 and R3 are independently a cyano group, a carboxy group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
X forms a phosphate group together with O that is bonded to X,
Y is a hydroxy group, and
Z is a hydrogen atom or a hydroxyl group;
wherein, in Formula III,
Ra is a cyano 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, 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 group, a naphthyl group, an indolyl group, a benzoimidazolyl group, a benzothiophenyl group, 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, and
Q2 is a hydrogen atom, and
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) is introduced into the second base sequence fraction as a base portion of the modified nucleotide having the structure corresponding to the monomer of Formula (II) or the amino acid analogue of the nucleotide having the structure corresponding to the monomer of Formula (III) by a phosphodiester bond, and
wherein the first oligonucleotide is at least partially complementary to the second oligonucleotide sequence fraction.

[claim45]
45. The method of hybridizing nucleic acid sequences according to claim 44,
wherein the first oligonucleotide contains the modified nucleotide having the structure corresponding to the monomer of Formula (I), and
wherein the modified nucleotide having the structure corresponding to the monomer of Formula (I) replaces at least one constituent nucleotide which is the photo-crosslinkable thymine (T) or uracil (U).

[claim46]
46. The method of hybridizing nucleic acid sequences according to claim 45, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.

[claim47]
47. The method of hybridizing nucleic acid sequences according to claim 45,
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, and R6 is a methyl group, and Q2 is a hydrogen atom.

[claim48]
48. The method of hybridizing nucleic acid sequences according to claim 44, wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom.

[claim49]
49. The method of hybridizing nucleic acid sequences according to claim 44,
wherein either:
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, and R6 is a methyl group, and Q2 is a hydrogen atom.

[claim50]
50. The method of hybridizing nucleic acid sequences according to claim 44,
wherein, in Formula (I), R is a cyano group, and Z is a hydrogen atom, and
wherein, either
1) in Formula (II), Ra is a cyano group, R2 and R3 are hydrogen atoms, and Z is a hydrogen atom, or
2) in Formula (III), Ra is a cyano group, R2, R3 and R4 are hydrogen atoms, R6 is a methyl group, and Q2is a hydrogen atom.
  • Inventor, and Inventor/Applicant
  • FUJIMOTO KENZO
  • NAKAMURA SHIGETAKA
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
ライセンスをご希望の方、特許の内容に興味を持たれた方は、問合せボタンを押してください。

PAGE TOP

close
close
close
close
close
close