JP6481161B2 - Method for photocleavage of photocrosslinking of photocrosslinked nucleic acid duplex - Google Patents
Method for photocleavage of photocrosslinking of photocrosslinked nucleic acid duplex Download PDFInfo
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- 238000000034 method Methods 0.000 title claims description 36
- 230000000295 complement effect Effects 0.000 claims description 35
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 claims description 18
- 125000003368 amide group Chemical group 0.000 claims description 16
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 16
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 16
- 239000001257 hydrogen Substances 0.000 claims description 16
- 229910052739 hydrogen Inorganic materials 0.000 claims description 16
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 13
- 125000006828 (C2-C7) alkoxycarbonyl group Chemical group 0.000 claims description 10
- 150000002431 hydrogen Chemical group 0.000 claims description 8
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 8
- 239000002777 nucleoside Substances 0.000 claims description 6
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 6
- 230000001737 promoting effect Effects 0.000 claims description 6
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- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000003776 cleavage reaction Methods 0.000 description 11
- 125000003729 nucleotide group Chemical group 0.000 description 9
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- 125000004453 alkoxycarbonyl group Chemical group 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000004132 cross linking Methods 0.000 description 6
- 238000013508 migration Methods 0.000 description 6
- 230000005012 migration Effects 0.000 description 6
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- 108020004414 DNA Proteins 0.000 description 5
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- 238000002474 experimental method Methods 0.000 description 5
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- 238000010438 heat treatment Methods 0.000 description 4
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- 239000012160 loading buffer Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- IHQKEDIOMGYHEB-UHFFFAOYSA-M sodium dimethylarsinate Chemical compound [Na+].C[As](C)([O-])=O IHQKEDIOMGYHEB-UHFFFAOYSA-M 0.000 description 3
- MUVQIIBPDFTEKM-QWWZWVQMSA-N (2r,3r)-2-aminobutane-1,3-diol Chemical group C[C@@H](O)[C@H](N)CO MUVQIIBPDFTEKM-QWWZWVQMSA-N 0.000 description 2
- MUVQIIBPDFTEKM-IMJSIDKUSA-N (2s,3s)-2-aminobutane-1,3-diol Chemical group C[C@H](O)[C@@H](N)CO MUVQIIBPDFTEKM-IMJSIDKUSA-N 0.000 description 2
- 125000004455 (C1-C3) alkylthio group Chemical group 0.000 description 2
- 125000006274 (C1-C3)alkoxy group Chemical group 0.000 description 2
- APQXWKHOGQFGTB-UHFFFAOYSA-N 1-ethenyl-9h-carbazole Chemical group C12=CC=CC=C2NC2=C1C=CC=C2C=C APQXWKHOGQFGTB-UHFFFAOYSA-N 0.000 description 2
- KJJPLEZQSCZCKE-UHFFFAOYSA-N 2-aminopropane-1,3-diol Chemical group OCC(N)CO KJJPLEZQSCZCKE-UHFFFAOYSA-N 0.000 description 2
- VIRDQWZTIAVLSE-UHFFFAOYSA-N 3-ethenyl-9h-carbazole Chemical group C1=CC=C2C3=CC(C=C)=CC=C3NC2=C1 VIRDQWZTIAVLSE-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 2
- 229910052731 fluorine Inorganic materials 0.000 description 2
- 125000001153 fluoro group Chemical group F* 0.000 description 2
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical group FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 125000001476 phosphono group Chemical group [H]OP(*)(=O)O[H] 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 2
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical group OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- USIRYNCTCFVRQR-IVZWLZJFSA-N 3-[1-[(2r,4s,5r)-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-2,4-dioxopyrimidin-5-yl]prop-2-enenitrile Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(C=CC#N)=C1 USIRYNCTCFVRQR-IVZWLZJFSA-N 0.000 description 1
- ICCMDPBNIJPGOH-FHWYHTKLSA-N C[C@@H](O)[C@@H](CO)NC(=O)CN1C2=C(C=CC=C2)C2=C1C=CC(\C=C\C#N)=C2 Chemical compound C[C@@H](O)[C@@H](CO)NC(=O)CN1C2=C(C=CC=C2)C2=C1C=CC(\C=C\C#N)=C2 ICCMDPBNIJPGOH-FHWYHTKLSA-N 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
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- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- PXBRQCKWGAHEHS-UHFFFAOYSA-N dichlorodifluoromethane Chemical compound FC(F)(Cl)Cl PXBRQCKWGAHEHS-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- 125000003835 nucleoside group Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
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Description
本発明は、光架橋核酸二重鎖の光架橋を光開裂させる方法に関する。 The present invention relates to a method for photocleaving a photocrosslink of a photocrosslinked nucleic acid duplex.
分子生物学的の分野において、核酸の連結と切断は、あらゆる応用の基礎となる重要な基本技術である。 In the field of molecular biology, ligation and cleavage of nucleic acids is an important basic technology that forms the basis of all applications.
光反応による核酸の連結の技術として、５−シアノビニルデオキシウリジンを使用した光連結技術（特許文献１：特許第３７５３９３８号、特許文献２：特許第３７５３９４２号）、３−ビニルカルバゾール構造を塩基部位に持つ修飾ヌクレオシド又はヌクレオシドアナログを使用した光架橋技術（特許文献３：特許第４８１４９０４号、特許文献４：特許第４９４０３１１号、特許文献５：国際公開ＷＯ２０１４／１５７５６５Ａ１号公報）がある。これらの技術によって核酸二重鎖の鎖間に光架橋を形成することができる。この光架橋は、共有結合による架橋であり、化学的に安定であるが、光反応によって再び開裂することができる。 As a technology for linking nucleic acids by photoreaction, photoligation technology using 5-cyanovinyldeoxyuridine (Patent Document 1: Patent No. 3753938, Patent Document 2: Patent No. 3753942), 3-vinylcarbazole structure as a base site There are photocrosslinking techniques using modified nucleosides or nucleoside analogs (Patent Document 3: Patent No. 4814904, Patent Document 4: Patent No. 4940311, Patent Document 5: International Publication WO 2014 / 157565A1). These techniques can form photocrosslinks between the strands of nucleic acid duplexes. This photocrosslinking is a covalent crosslink and is chemically stable, but can be cleaved again by a photoreaction.
上記のように、３−ビニルカルバゾール構造を塩基部位に持つ修飾ヌクレオシドによって、核酸二重鎖の鎖間に形成された光架橋は、光反応による開裂（光開裂）が可能である。しかし、核酸二重鎖の鎖間に形成された光架橋の開裂を、十分に効率よく進行させるためには、加熱条件下（例えば９０℃）で、短波長（例えば３１２ｎｍ）の光照射を行うことが必要であった。このような加熱や短波長の光は細胞にとって好ましいものではない。 As described above, the photocrosslinking formed between the strands of the nucleic acid duplex by the modified nucleoside having a 3-vinylcarbazole structure at the base site can be cleaved by photoreaction (photocleavage). However, in order to allow the cleavage of the photocrosslink formed between the nucleic acid duplex strands to proceed sufficiently efficiently, light irradiation with a short wavelength (eg 312 nm) is performed under heating conditions (eg 90 ° C.). It was necessary. Such heating and short-wavelength light are not preferable for cells.
したがって、本発明の目的は、従来よりも低温で、従来よりも長波長の光照射によって、核酸二重鎖の鎖間に形成された光架橋を、高い効率で開裂させる方法を提供することにある。 Therefore, an object of the present invention is to provide a method for efficiently cleaving a photocrosslink formed between strands of a nucleic acid duplex by irradiation with light having a longer wavelength than that of a conventional one at a lower temperature. is there.
本発明者は、上記の光応答性修飾核酸によって形成された二重鎖内の光架橋の光開裂について、鋭意研究してきたところ、二重鎖の末端に一重鎖部分を設けて、該末端の一重鎖部分を有する鎖の一重鎖部分と二重鎖部分に対して相補的な配列を有する挿入核酸の鎖を、光架橋核酸二重鎖と溶液中で接触可能に添加して、光照射すると、従来よりも低温で、従来よりも長波長の光照射によっても、光開裂が高い効率で進行することを見いだして、本発明に到達した。 The present inventor has intensively studied the photocleavage of the photocrosslink in the duplex formed by the above-mentioned photoresponsive modified nucleic acid. As a result, a single-stranded portion was provided at the end of the duplex, When an inserted nucleic acid strand having a sequence complementary to a single-stranded portion and a double-stranded portion having a single-stranded portion is added so as to be able to come into contact with a photocross-linked nucleic acid duplex in solution, and then irradiated with light The inventors have found that photocleavage proceeds with high efficiency even when irradiated with light having a wavelength lower than that of the prior art and at a wavelength longer than that of the prior art.
したがって、本発明は次の（１）以下にもある。
（１）
核酸二重鎖内の鎖間に光応答性塩基によって形成された光架橋を有する、光架橋核酸二重鎖に対して、光照射して、光架橋を光開裂させる方法であって、
光架橋核酸二重鎖は、光架橋性のビニル構造を有する光応答性塩基を有する光応答性修飾核酸の鎖と、該光応答性修飾核酸に対して相補的な配列を有する被光架橋性核酸の鎖とによって二重鎖形成され、
光応答性修飾核酸の鎖と被光架橋性核酸の鎖とは、光応答性塩基によって光架橋され、
光架橋核酸二重鎖は、二重鎖の末端に一重鎖部分を有しており、
該末端の一重鎖部分を有する鎖の一重鎖部分と二重鎖部分に対して相補的な配列を有する挿入核酸の鎖を、光架橋核酸二重鎖と溶液中で接触可能に添加して、光照射する工程、
を含む、方法。
（２）
光架橋性のビニル構造を有する光応答性塩基が、光架橋性のビニルカルバゾール構造を有する光応答性塩基である、（１）に記載の方法。
（３）
光架橋性のビニル構造を有する光応答性塩基を有する光応答性修飾核酸が、次の式（Ｉ）：
Ｒ１２及びＲ１３は、それぞれ独立に、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、又は水素であり、
Ｒ４は、水素を表し、
Ｒ５は、水酸基を表し、
Ｒ６は、水素又は水酸基である）
で表される修飾ヌクレオシドが、光架橋性のビニル構造を有する光応答性塩基として、リン酸ジエステル結合によって塩基配列中に導入されてなる修飾核酸であり、
光応答性塩基によって形成された光架橋が、
光応答性修飾核酸の塩基配列中の光架橋性のビニル構造を有する光応答性塩基と、
被光架橋性核酸の塩基配列中のＴ又はＵとの間に形成された光架橋である、（１）に記載の方法。
（４）
光架橋性のビニル構造を有する光応答性塩基を有する光応答性修飾核酸が、次の式（ＩＩ）：
Ｒ１１は、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、ホスホノ基、スルホ基、又は水素原子を表し、
Ｒ１２及びＲ１３は、それぞれ独立に、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、又は水素原子を表し、
Ｒ７は、水素原子、水酸基、Ｃ１〜Ｃ３のアルコキシ基、Ｃ１〜Ｃ３のアルキルスルファニル基、ニトロ基、フッ素原子、フッ化メチル基、Ｃ６〜Ｃ１２の単環式又は二環式の芳香族化合物の１価基、Ｃ６〜Ｃ１２の単環式又は二環式の複素環系芳香族化合物の１価基、又は、次の式：
で表される１価基を表し、
Ｒ８は、水素原子、メチル基、又はエチル基を表し、
Ｑ1は、式ＩＩＩでＱ1に結合しているＯと一体となって形成されたリン酸基を表し、
Ｑ2は、水素原子を表す）
で表される修飾ヌクレオチドの塩基部分として、上記修飾ヌクレオチドのリン酸ジエステル結合によって光応答性部分塩基配列中に導入されている、（１）〜（３）のいずれかに記載の方法。
（５）
式ＩＩ中において、次の式（ＩＩａ）：
次の式：
次の式：
又は、次の式：
（６）
末端の一重鎖部分を有する鎖が、光応答性修飾核酸の鎖、又は挿入核酸の鎖であり、
該末端の一重鎖部分が、光応答性修飾核酸の鎖の３’末端又は５’末端、または挿入核酸の鎖の３’末端又は５’末端である、（１）〜（５）のいずれかに記載の方法。
（７）
光架橋核酸二重鎖の末端の一重鎖部分が、１〜３０塩基の塩基長である、（１）〜（６）のいずれかに記載の方法。
（８）
末端の一重鎖部分を有する鎖の一重鎖部分と二重鎖部分に対して相補的な配列を有する挿入核酸の鎖が、一重鎖、又は一重鎖部分を有する二重鎖である、（１）〜（７）のいずれかに記載の方法。
（９）
末端の一重鎖部分を有する鎖が、光応答性修飾核酸の鎖であり、
末端の一重鎖部分が、光応答性修飾核酸の鎖の３’末端又は５’末端であり、
挿入核酸の鎖が、光応答性修飾核酸に対して相補的な配列を有する一重鎖である、（１）〜（８）のいずれかに記載の方法。
（１０）
光照射が、３１２〜４００ｎｍの範囲の波長の光照射によって行われる、（１）〜（９）のいずれかに記載の方法。
（１１）
光照射が、１〜６０℃の範囲の温度において行われる、（１）〜（１０）のいずれかに記載の方法。
（１２）
光照射が、４〜４０℃の範囲の温度において行われる、（１）〜（１０）のいずれかに記載の方法。
（１３）
（１）〜（１２）のいずれかに記載の方法によって、
核酸二重鎖内の鎖間の光架橋を光開裂させて、光架橋されていない光応答性修飾核酸を、製造する方法。
（１４）
（１）〜（１２）のいずれかに記載の方法によって、
核酸二重鎖内の鎖間の光架橋を光開裂させて、光架橋されていない被光架橋性核酸を、製造する方法。
（１５）
（１）〜（１２）のいずれかに記載の挿入核酸からなる、光架橋核酸二重鎖用光開裂促進剤。
（１６）
（１）〜（１２）のいずれかに記載の挿入核酸の、光架橋核酸二重鎖内光開裂促進のための使用。
Therefore, the present invention is also the following (1) and below.
(1)
A method of photoirradiating a photocrosslinked nucleic acid duplex by photoirradiating a photocrosslinked nucleic acid duplex having a photocrosslink formed by a photoresponsive base between strands in a nucleic acid duplex,
The photocrosslinkable nucleic acid duplex is a photocrosslinkable nucleic acid having a photoresponsive base having a photocrosslinkable vinyl structure and a sequence complementary to the photoresponsive base. Double-stranded with the nucleic acid strand,
The strand of the photoresponsive modified nucleic acid and the photocrosslinkable nucleic acid strand are photocrosslinked by a photoresponsive base,
The photocrosslinking nucleic acid duplex has a single-stranded portion at the end of the duplex,
A strand of an inserted nucleic acid having a sequence complementary to the single-stranded portion and double-stranded portion of the strand having the single-stranded portion of the terminal is added in contact with the photocrosslinking nucleic acid duplex in solution; A light irradiation step,
Including a method.
(2)
The method according to (1), wherein the photoresponsive base having a photocrosslinkable vinyl structure is a photoresponsive base having a photocrosslinkable vinylcarbazole structure.
(3)
A photoresponsive modified nucleic acid having a photoresponsive base having a photocrosslinkable vinyl structure is represented by the following formula (I):
R12 and R13 are each independently a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen;
R4 represents hydrogen,
R5 represents a hydroxyl group,
R6 is hydrogen or a hydroxyl group)
The modified nucleoside represented by is a modified nucleic acid introduced into a base sequence by a phosphodiester bond as a photoresponsive base having a photocrosslinkable vinyl structure,
The photocrosslink formed by the photoresponsive base is
A photoresponsive base having a photocrosslinkable vinyl structure in the base sequence of the photoresponsive modified nucleic acid;
The method according to (1), wherein the method is photocrosslinking formed with T or U in the base sequence of the photocrosslinkable nucleic acid.
(4)
A photoresponsive modified nucleic acid having a photoresponsive base having a photocrosslinkable vinyl structure is represented by the following formula (II):
R11 represents a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, a phosphono group, a sulfo group, or a hydrogen atom,
R12 and R13 each independently represent a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R7 is a hydrogen atom, a hydroxyl group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a methyl fluoride group, or a C6-C12 monocyclic or bicyclic aromatic compound. A monovalent group, a monovalent group of a C6-C12 monocyclic or bicyclic heterocyclic aromatic compound, or the following formula:
Represents a monovalent group represented by:
R8 represents a hydrogen atom, a methyl group, or an ethyl group,
Q 1 represents a phosphate group formed integrally with O bonded to Q 1 in Formula III;
Q 2 represents a hydrogen atom)
The method according to any one of (1) to (3), wherein the base moiety of the modified nucleotide represented by the formula is introduced into the photoresponsive partial base sequence by a phosphodiester bond of the modified nucleotide.
(5)
In formula II, the following formula (IIa):
The following formula:
The following formula:
Or the following formula:
(6)
The strand having the terminal single-stranded portion is a strand of a light-responsive modified nucleic acid or a strand of an inserted nucleic acid;
Any of (1) to (5), wherein the single-stranded part of the terminal is the 3 ′ end or 5 ′ end of the strand of the light-responsive modified nucleic acid, or the 3 ′ end or 5 ′ end of the strand of the inserted nucleic acid. The method described in 1.
(7)
The method according to any one of (1) to (6), wherein the single-stranded portion at the end of the photocrosslinked nucleic acid duplex has a base length of 1 to 30 bases.
(8)
The strand of the inserted nucleic acid having a sequence complementary to the single-stranded portion and the double-stranded portion of the strand having the terminal single-stranded portion is a single strand or a double-stranded portion having a single-stranded portion, (1) -The method in any one of (7).
(9)
The strand having the terminal single-stranded portion is a strand of a light-responsive modified nucleic acid,
The single-stranded part of the terminal is the 3 ′ end or 5 ′ end of the strand of the light-responsive modified nucleic acid,
The method according to any one of (1) to (8), wherein the strand of the inserted nucleic acid is a single strand having a sequence complementary to the light-responsive modified nucleic acid.
(10)
The method according to any one of (1) to (9), wherein the light irradiation is performed by light irradiation with a wavelength in the range of 312 to 400 nm.
(11)
The method according to any one of (1) to (10), wherein the light irradiation is performed at a temperature in the range of 1 to 60 ° C.
(12)
The method according to any one of (1) to (10), wherein the light irradiation is performed at a temperature in the range of 4 to 40 ° C.
(13)
By the method according to any one of (1) to (12),
A method for producing a photoresponsive modified nucleic acid that is not photocrosslinked by photocleaving photocrosslinks between strands in a nucleic acid duplex.
(14)
By the method according to any one of (1) to (12),
A method for producing a photocrosslinkable nucleic acid that is not photocrosslinked by photocleaving photocrosslinks between strands in a nucleic acid duplex.
(15)
A photocleavage accelerator for photocrosslinked nucleic acid duplexes, comprising the inserted nucleic acid according to any one of (1) to (12).
(16)
Use of the inserted nucleic acid according to any one of (1) to (12) for promoting photocleavage in a photocrosslinked nucleic acid duplex.
本発明によれば、従来よりも低温で、従来よりも長波長の光照射によっても、光開裂を高い効率で進行させることができる。そのために、生体分子に穏和な条件で光開裂が可能であり、あるいは細胞内や生体内で光開裂することも可能となる。 According to the present invention, photocleavage can proceed with high efficiency even by irradiation with light having a longer wavelength than that of the prior art at a temperature lower than that of the prior art. Therefore, photocleavage can be performed under conditions that are moderate to biomolecules, or photocleavage can be performed in cells or in vivo.
具体的な実施の形態をあげて、以下に本発明を詳細に説明する。本発明は、以下にあげる具体的な実施他の形態に限定されるものではない。 The present invention will be described in detail below by giving specific embodiments. The present invention is not limited to the following specific embodiments and other embodiments.
［光架橋の光開裂］
本発明は、核酸二重鎖内の鎖間に光応答性塩基によって形成された光架橋を有する、光架橋核酸二重鎖に対して、光照射して、光架橋を光開裂させる方法であって、光架橋核酸二重鎖は、光架橋性のビニル構造を有する光応答性塩基を有する光応答性修飾核酸の鎖と、該光応答性修飾核酸に対して相補的な配列を有する被光架橋性核酸の鎖とによって二重鎖形成され、光応答性修飾核酸の鎖と被光架橋性核酸の鎖とは、光応答性塩基によって光架橋され、光架橋核酸二重鎖は、二重鎖の末端に一重鎖部分を有しているとしたうえで、該末端の一重鎖部分を有する鎖の一重鎖部分と二重鎖部分に対して相補的な配列を有する挿入核酸の鎖を、光架橋核酸二重鎖と溶液中で接触可能に添加して、光照射する工程を行うことによって実施される。
[Photocleavage of photocrosslinking]
The present invention is a method of photoirradiating a photocrosslinked nucleic acid duplex having a photocrosslink formed by a photoresponsive base between strands in the nucleic acid duplex to photocleavage the photocrosslink. The photo-crosslinking nucleic acid duplex is a photo-responsive modified nucleic acid strand having a photo-responsive base having a photo-crosslinkable vinyl structure and a light-receiving sequence having a sequence complementary to the photo-responsive modified nucleic acid. The strand of the crosslinkable nucleic acid is double-stranded, the photoresponsive modified nucleic acid strand and the photocrosslinkable nucleic acid strand are photocrosslinked by a photoresponsive base, and the photocrosslinked nucleic acid duplex is double-stranded. A strand of an inserted nucleic acid having a single-stranded portion at the end of the strand and having a sequence complementary to the single-stranded portion and double-stranded portion of the strand having the single-stranded portion at the end, It is carried out by adding a photocrosslinkable nucleic acid duplex so as to be contactable in a solution and performing a light irradiation step.
好適な実施の態様において、光架橋性のビニル構造を有する光応答性塩基は、光架橋性のビニルカルバゾール構造を有する光応答性塩基である。 In a preferred embodiment, the photoresponsive base having a photocrosslinkable vinyl structure is a photoresponsive base having a photocrosslinkable vinylcarbazole structure.
本発明によれば、光架橋された核酸二重鎖を光開裂するために光照射する際に、上記挿入核酸を、添加することによって光開裂反応を促進して、従来よりも低温で且つ長波長の光照射によっても、高い効率で光開裂された核酸を得ることができる。 According to the present invention, when the photo-crosslinked nucleic acid duplex is irradiated with light for photocleavage, the insertion nucleic acid is added to promote the photocleavage reaction so that the photocleavage reaction is performed at a lower temperature and longer than before. Also by irradiation with light of a wavelength, a nucleic acid that has been photo-cleaved with high efficiency can be obtained.
本発明によるこの優れた効果が生じるメカニズムについて、本発明者は、鎖交換反応が関与していると考えている。図１に示すような光架橋の形成は、光照射によって逆反応を進行させることが知られている。従来は、３１２ｎｍといった紫外領域の光照射を加熱下（例えば９０℃）で行うことが、光開裂の効率の観点から必要とされていた。このような条件では、生体内や細胞内での光開裂の実施は、現実的には行えない。ところが、本発明による挿入核酸の添加を行うと、挿入核酸の鎖が、光架橋核酸二重鎖の末端の一重鎖部分（sticky end）に付着して、図２に示すような鎖交換反応が進行すると考えられる。そして、低温で長波長の光照射によって光開裂が生じた場合に、鎖交換が行われる結果、光開裂反応の逆反応が生じるチャンスを極めて低減することによって、光反応を一方向に進行させて、上記の優れた効果を実現すると考えられる。したがって、以下の具体的な実施の態様に限られずに、上記挿入核酸による鎖交換反応によって、光架橋された核酸二重鎖の光開裂を促進する技術は、本発明の範囲内にある。 The present inventor believes that a chain exchange reaction is involved in the mechanism by which this excellent effect of the present invention occurs. The formation of photocrosslinking as shown in FIG. 1 is known to cause a reverse reaction by light irradiation. Conventionally, it has been necessary from the viewpoint of photocleavage efficiency to perform irradiation of light in the ultraviolet region of 312 nm under heating (for example, 90 ° C.). Under such conditions, photocleavage in vivo or in cells cannot be performed practically. However, when the inserted nucleic acid according to the present invention is added, the strand of the inserted nucleic acid is attached to the single-stranded portion (sticky end) of the end of the photocrosslinked nucleic acid duplex, and the strand exchange reaction as shown in FIG. It is thought to progress. Then, when photocleavage occurs due to light irradiation at a low wavelength at a low temperature, as a result of chain exchange, the chance of the reverse reaction of the photocleavage reaction is greatly reduced, so that the photoreaction proceeds in one direction. It is considered that the above excellent effect is realized. Therefore, the present invention is not limited to the following specific embodiments, and a technique for promoting photocleavage of a photocrosslinked nucleic acid duplex by a strand exchange reaction with the inserted nucleic acid is within the scope of the present invention.
［光架橋核酸二重鎖］
光架橋核酸二重鎖は、光架橋性のビニル構造を有する光応答性塩基を有する光応答性修飾核酸の鎖と、該光応答性修飾核酸に対して相補的な配列を有する被光架橋性核酸の鎖とによって二重鎖形成されている。光架橋核酸二重鎖は、本発明の実施が可能である限り、さらに修飾されていてもよく、例えば、いずれかの鎖が修飾されて、検出や定量のための標識部位が設けられていてもよく、例えば、いずれかの鎖が修飾されて、ビーズやプレートなどの基材や担体に固定されていてもよい。
[Photocrosslinked nucleic acid duplex]
The photocrosslinkable nucleic acid duplex is a photocrosslinkable nucleic acid having a photoresponsive base having a photocrosslinkable vinyl structure and a sequence complementary to the photoresponsive base. Double strands are formed by nucleic acid strands. The photocrosslinked nucleic acid duplex may be further modified as long as the present invention can be carried out. For example, either strand is modified to provide a labeling site for detection or quantification. For example, any one of the chains may be modified and fixed to a substrate or carrier such as a bead or a plate.
光架橋核酸二重鎖の二重鎖部分の塩基長は、本発明の実施が可能である限り、特に制限はないが、例えば、１０〜１００００塩基、２０〜１０００塩基、２０〜１００塩基の塩基長とすることができる。 Although the base length of the double-stranded part of the photocrosslinking nucleic acid duplex is not particularly limited as long as the present invention can be carried out, for example, 10 to 10,000 bases, 20 to 1000 bases, 20 to 100 bases Can be long.
［末端の一重鎖部分］
光架橋核酸二重鎖は、二重鎖の末端に一重鎖部分を有する。この末端の一重鎖部分(sticky end)に、挿入核酸が付着して、鎖交換反応が進行すると考えられる。そのため、この末端の一重鎖部分は、本発明の実施が可能である限り、二重鎖のいずれの鎖の末端に存在していてもよく、３’末端又は５’末端のいずれの末端に存在していてもよい。例えば、光応答性修飾核酸の鎖の３’末端、光応答性修飾核酸の鎖の５’末端、被光架橋性核酸の鎖の３’末端、又は被光架橋性核酸の鎖の５’末端に存在するものとできる。
[Single-stranded part of terminal]
Photocrosslinked nucleic acid duplexes have a single-stranded portion at the end of the duplex. It is thought that the inserted nucleic acid is attached to the single-stranded portion (sticky end) of this end, and the strand exchange reaction proceeds. Therefore, the single-stranded part of this end may be present at the end of any strand of the duplex as long as the present invention can be carried out, and is present at any end of the 3 ′ end or 5 ′ end. You may do it. For example, the 3 ′ end of the strand of the photoresponsive modified nucleic acid, the 5 ′ end of the strand of the photoresponsive modified nucleic acid, the 3 ′ end of the strand of the photocrosslinkable nucleic acid, or the 5 ′ end of the strand of the photocrosslinkable nucleic acid Can exist.
末端の一重鎖部分の塩基長は、本発明の実施が可能である限り、特に制限はないが、例えば、１〜３０塩基、１〜２０塩基、１〜１０塩基、３〜１０塩基、４〜１０塩基、６〜１０塩基、４〜８塩基、６〜８塩基の塩基長とすることができる。 The base length of the terminal single-stranded portion is not particularly limited as long as the present invention can be carried out, but for example, 1 to 30 bases, 1 to 20 bases, 1 to 10 bases, 3 to 10 bases, 4 to 4 The base length can be 10 bases, 6-10 bases, 4-8 bases, 6-8 bases.
［光架橋］
光架橋核酸二重鎖では、光応答性修飾核酸の鎖と被光架橋性核酸の鎖とが、光架橋性のビニル構造を有する光応答性塩基の光反応によってあらかじめ光架橋されている。好適な実施の態様において、光架橋は、光応答性修飾核酸の塩基配列中の光架橋性のビニル構造を有する光応答性塩基と、被光架橋性核酸の塩基配列中のＴ又はＵとの間に形成された光架橋である。この光架橋は、公知の手段によって形成することができる。
[Photocrosslinking]
In the photocrosslinked nucleic acid duplex, the chain of the photoresponsive modified nucleic acid and the chain of the photocrosslinkable nucleic acid are photocrosslinked in advance by a photoreaction of a photoresponsive base having a photocrosslinkable vinyl structure. In a preferred embodiment, the photocrosslinking is performed between the photoresponsive base having a photocrosslinkable vinyl structure in the base sequence of the photoresponsive modified nucleic acid and T or U in the base sequence of the photocrosslinkable nucleic acid. It is a photocrosslinking formed between them. This photocrosslinking can be formed by known means.
光架橋された塩基から、末端の一重鎖部分の塩基までの距離は、本発明の実施が可能である限り、特に制限はないが、例えば、１〜５０塩基、２〜１０塩基の塩基長とすることができる。 The distance from the photocrosslinked base to the base of the single-stranded portion at the end is not particularly limited as long as the present invention can be carried out. For example, the base length is 1 to 50 bases and 2 to 10 bases. can do.
［光応答性修飾核酸］
好適な実施の態様において、光応答性修飾核酸は、次の式（Ｉ）：
In a preferred embodiment, the light-responsive modified nucleic acid has the following formula (I):
式Ｉにおいて、Ｒ１１は、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、又は水素を表し、
Ｒ１２及びＲ１３は、それぞれ独立に、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、又は水素を表し、
Ｒ４は、水素を表し、
Ｒ５は、水酸基を表し、
Ｒ６は、水素又は水酸基を表す。
In Formula I, R11 represents a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen,
R12 and R13 each independently represent a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen,
R4 represents hydrogen,
R5 represents a hydroxyl group,
R6 represents hydrogen or a hydroxyl group.
好適な実施の態様において、式ＩのＲ１１は、シアノ基、アミド基、カルボキシル基、アルコキシカルボニル基、又は水素であり、好ましくは、シアノ基、アミド基、カルボキシル基、アルコキシカルボニル基、又は水素であり、さらに好ましくは、シアノ基、アミド基、カルボキシル基、又はアルコキシカルボニル基である。アルコキシカルボニル基は、好ましくはＣ２〜Ｃ７、さらに好ましくはＣ２〜Ｃ６、さらに好ましくはＣ２〜Ｃ５、さらに好ましくはＣ２〜Ｃ４、さらに好ましくはＣ２〜Ｃ３、特に好ましくはＣ２のものを使用することができる。 In a preferred embodiment, R11 of formula I is a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group, or hydrogen, preferably a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group, or hydrogen. And more preferably a cyano group, an amide group, a carboxyl group, or an alkoxycarbonyl group. The alkoxycarbonyl group is preferably C2 to C7, more preferably C2 to C6, more preferably C2 to C5, further preferably C2 to C4, further preferably C2 to C3, and particularly preferably C2. it can.
好適な実施の態様において、式ＩのＲ１２及びＲ１３は、それぞれ独立に、シアノ基、アミド基、カルボキシル基、アルコキシカルボニル基、又は水素であり、好ましくは、シアノ基、アミド基、カルボキシル基、アルコキシカルボニル基、又は水素であり、さらに好ましくは、シアノ基、アミド基、カルボキシル基、又はアルコキシカルボニル基である。アルコキシカルボニル基は、好ましくはＣ２〜Ｃ７、さらに好ましくはＣ２〜Ｃ６、さらに好ましくはＣ２〜Ｃ５、さらに好ましくはＣ２〜Ｃ４、さらに好ましくはＣ２〜Ｃ３、特に好ましくはＣ２のものを使用することができる。 In a preferred embodiment, R12 and R13 of formula I are each independently a cyano group, an amide group, a carboxyl group, an alkoxycarbonyl group, or hydrogen, preferably a cyano group, an amide group, a carboxyl group, an alkoxy A carbonyl group or hydrogen, more preferably a cyano group, an amide group, a carboxyl group, or an alkoxycarbonyl group. The alkoxycarbonyl group is preferably C2 to C7, more preferably C2 to C6, more preferably C2 to C5, further preferably C2 to C4, further preferably C2 to C3, and particularly preferably C2. it can.
好適な実施の態様において、光応答性修飾核酸は、次の式（ＩＩ）：
式ＩＩにおいて、
Ｒ１１は、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、ホスホノ基、スルホ基、又は水素原子を表し、
Ｒ１２及びＲ１３は、それぞれ独立に、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、又は水素原子を表し、
Ｒ７は、水素原子、水酸基、Ｃ１〜Ｃ３のアルコキシ基、Ｃ１〜Ｃ３のアルキルスルファニル基、ニトロ基、フッ素原子、フッ化メチル基、Ｃ６〜Ｃ１２の単環式又は二環式の芳香族化合物の１価基、Ｃ６〜Ｃ１２の単環式又は二環式の複素環系芳香族化合物の１価基、又は、次の式：
で表される１価基を表し、
Ｒ８は、水素原子、メチル基、又はエチル基を表し、
Ｑ1は、式ＩＩでＱ1に結合しているＯと一体となって形成されたリン酸基を表し、
Ｑ2は、水素原子を表す。
式ＩＩにおけるＲ１１、Ｒ１２及びＲ１３は、それぞれ独立に、式Ｉについて上述したＲ１１、Ｒ１２及びＲ１３とすることができる。
In Formula II,
R11 represents a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, a phosphono group, a sulfo group, or a hydrogen atom,
R12 and R13 each independently represent a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or a hydrogen atom,
R7 is a hydrogen atom, a hydroxyl group, a C1-C3 alkoxy group, a C1-C3 alkylsulfanyl group, a nitro group, a fluorine atom, a methyl fluoride group, or a C6-C12 monocyclic or bicyclic aromatic compound. A monovalent group, a monovalent group of a C6-C12 monocyclic or bicyclic heterocyclic aromatic compound, or the following formula:
Represents a monovalent group represented by:
R8 represents a hydrogen atom, a methyl group, or an ethyl group,
Q 1 represents a phosphate group formed integrally with O bonded to Q 1 in Formula II;
Q 2 represents a hydrogen atom.
R11, R12, and R13 in Formula II can each independently be R11, R12, and R13 described above for Formula I.
例えば、式ＩＩのＲ７として、次に挙げる基を使用することができる。（ただし、波線は遊離原子価の位置を示している。） For example, the following groups can be used as R7 in Formula II. (However, the wavy line indicates the position of the free valence.)
好適な実施の態様において、式ＩＩ中において、次の式（ＩＩａ）：
次の式：
次の式：
又は、次の式：
The following formula:
The following formula:
Or the following formula:
上記式ＩＩで表される修飾ヌクレオチドは、天然のヌクレオチドや式Ｉの修飾ヌクレオチドであればリボース（又はデオキシリボース）構造の糖骨格である部分が、上記式ＩＩａで表される骨格構造で代替されたものとなっている。そこで、式ＩＩで表される修飾ヌクレオチドは、光応答性人工ヌクレオチドアナログということもできる。驚くべきことに、式ＩＩの修飾ヌクレオチドは、このような骨格構造の相違にも関わらず、核酸への組み込みと光応答性に関しては、光応答性塩基として振る舞う。本発明者はこの発見に基づいて、すでに日本国特許出願（特願２０１３−７０３８１）を行った。 In the modified nucleotide represented by the above formula II, in the case of a natural nucleotide or a modified nucleotide of the formula I, the sugar skeleton portion of the ribose (or deoxyribose) structure is replaced by the skeleton structure represented by the above formula IIa. It has become. Therefore, the modified nucleotide represented by Formula II can also be referred to as a photoresponsive artificial nucleotide analog. Surprisingly, the modified nucleotide of Formula II behaves as a photoresponsive base with respect to incorporation into nucleic acids and photoresponsiveness despite such differences in backbone structure. Based on this discovery, the present inventor has already filed a Japanese patent application (Japanese Patent Application No. 2013-70381).
好適な実施の態様において、上記式ＩＩで表される修飾ヌクレオチドは、配列中に、次の式で表されるｃｎｖＤを、光応答性修飾塩基を有するヌクレオシドとして、リン酸ジエステル結合して有している。
［被光架橋性核酸］
被光架橋性核酸は、光応答性修飾核酸に対して相補的な塩基配列を有しており、この相補性によって二重鎖を形成している。光応答性修飾核酸に対する相補性は、二重鎖を形成して本発明を実施できる範囲内であればよい。特に、光応答性修飾核酸の塩基配列中の光架橋性のビニル構造を有する光応答性塩基は、いわゆる水素結合による塩基対を形成しないので、被光架橋性核酸の塩基配列中において光応答性塩基と相補的な位置にある塩基は、いずれの塩基であってもよい。例えば、被光架橋性核酸は、光架橋核酸二重鎖の二重鎖部分において、光応答性修飾核酸に対して、９０％以上、９４％以上、９５％以上、９６％以上、９８％以上、９９％以上相補的な塩基配列とすることができる。好適な実施の態様において、被光架橋性核酸は、被光架橋性核酸の塩基配列中において光応答性塩基と相補的な位置にある塩基を除いて、光架橋核酸二重鎖の二重鎖部分において光応答性塩基に対して完全に相補的な塩基配列とすることができる。
[Photocrosslinkable nucleic acid]
The photocrosslinkable nucleic acid has a base sequence complementary to the light-responsive modified nucleic acid, and forms a duplex by this complementarity. Complementarity to the light-responsive modified nucleic acid may be within a range in which the present invention can be carried out by forming a duplex. In particular, photoresponsive bases having a photocrosslinkable vinyl structure in the base sequence of the photoresponsive modified nucleic acid do not form so-called hydrogen-bonded base pairs, and therefore are photoresponsive in the base sequence of the photocrosslinkable nucleic acid. The base at a position complementary to the base may be any base. For example, the photocrosslinkable nucleic acid is 90% or more, 94% or more, 95% or more, 96% or more, 98% or more of the photoresponsive modified nucleic acid in the double-stranded part of the photocrosslinking nucleic acid duplex. 99% or more of the complementary nucleotide sequence. In a preferred embodiment, the photocrosslinkable nucleic acid is a duplex of a photocrosslinkable nucleic acid duplex except for a base that is complementary to the photoresponsive base in the base sequence of the photocrosslinkable nucleic acid. The base sequence can be completely complementary to the photoresponsive base in the portion.
［挿入核酸］
挿入核酸は、光架橋核酸二重鎖の末端の一重鎖部分を有する鎖の一重鎖部分(sticky end)に対して相補的な塩基配列を有し、さらに連続してその一重鎖部分を有する鎖の二重鎖部分に対しても相補的な塩基配列を有する。このような相補的な塩基配列によって、光架橋核酸二重鎖の末端の一重鎖部分(sticky end)に付着した挿入核酸が、鎖交換反応を行って、光開裂部位に達して、光開裂反応を促進するものと考えられる。そこで、挿入核酸は、一重鎖部分を有する鎖の二重鎖部分に対して、光架橋核酸二重鎖のもう一方の鎖と比較して、同程度以上に高い相補性を有する塩基配列とすることが好ましい。
[Inserted nucleic acid]
The inserted nucleic acid has a base sequence complementary to the single-stranded portion (sticky end) of the strand having the single-stranded portion at the end of the photocross-linked nucleic acid duplex, and further has the single-stranded portion in succession. It also has a complementary base sequence to the double-stranded part. With such a complementary base sequence, the inserted nucleic acid attached to the single-stranded portion (sticky end) of the photocrosslinking nucleic acid duplex undergoes a strand exchange reaction, reaches the photocleavage site, and undergoes a photocleavage reaction. It is thought that it promotes. Therefore, the inserted nucleic acid has a base sequence having a higher degree of complementarity than the other strand of the photocrosslinked nucleic acid duplex with respect to the duplex portion of the strand having a single-stranded portion. It is preferable.
例えば、光架橋核酸二重鎖の末端の一重鎖部分を有する鎖が、光応答性修飾核酸である場合には、挿入核酸は、一重鎖部分に対して相補的な塩基配列を有し、さらに連続して光応答性修飾核酸の二重鎖部分の鎖に対して相補的な塩基配列を有し、特に、被光架橋性核酸の鎖よりも、光応答性修飾核酸の二重鎖部分の鎖に対して、同じ又はより高い割合で相補的な塩基配列を有していることが好ましい。 For example, when the strand having the single-stranded portion at the end of the photocrosslinking nucleic acid duplex is a photoresponsive modified nucleic acid, the inserted nucleic acid has a base sequence complementary to the single-stranded portion, and It has a base sequence complementary to the strand of the double-stranded portion of the light-responsive modified nucleic acid successively, and in particular, the double-stranded portion of the photo-responsive modified nucleic acid rather than the strand of the photo-crosslinkable nucleic acid. It is preferable to have a complementary base sequence at the same or higher ratio to the strand.
好適な実施の態様において、挿入核酸の塩基長は、光架橋核酸二重鎖の末端の一重鎖部分を有する鎖と同じ塩基長であり、例えば、光応答性修飾核酸又は被光架橋性核酸と同じ塩基長とすることができる。 In a preferred embodiment, the base length of the inserted nucleic acid is the same base length as the strand having the single-stranded portion at the end of the photocrosslinked nucleic acid duplex, for example, the photoresponsive modified nucleic acid or the photocrosslinkable nucleic acid The same base length can be used.
好適な実施の態様において、挿入核酸の鎖は、一重鎖として添加される。しかし、一重鎖を生成可能な二重鎖の形態で添加することもできる。挿入核酸の鎖の中で、光架橋核酸二重鎖の末端の一重鎖部分に対する相補的な鎖が一重鎖として露出して、鎖交換反応が進行可能な二重鎖の形態で、挿入核酸の鎖を添加してもよい。 In a preferred embodiment, the strand of inserted nucleic acid is added as a single strand. However, it can also be added in the form of a double chain capable of producing a single chain. In the inserted nucleic acid strand, the complementary strand to the single-stranded portion at the end of the photocrosslinked nucleic acid duplex is exposed as a single strand, and in the form of a duplex that allows the strand exchange reaction to proceed, Chains may be added.
［光照射］
本発明による光開裂の促進は、広範な波長範囲において実現されるために、光照射は、光開裂が可能な波長であれば、特に制限なく使用できる。例えば、３００〜４００ｎｍ、３１２〜４００ｎｍ、３２０〜４００ｎｍの波長を使用できる。本発明は、従来は光開裂の効率が低かった長波長（例えば３６６ｎｍ）の光照射によっても、十分に高い効率となるまで光開裂を促進できる点で、特に優れている。
[Light irradiation]
Since the promotion of photocleavage according to the present invention is realized in a wide wavelength range, light irradiation can be used without particular limitation as long as it is a wavelength capable of photocleavage. For example, wavelengths of 300 to 400 nm, 312 to 400 nm, and 320 to 400 nm can be used. The present invention is particularly excellent in that photocleavage can be accelerated to sufficiently high efficiency even by irradiation with light having a long wavelength (for example, 366 nm), which has conventionally had low photocleavage efficiency.
本発明による光開裂の促進は、広範な温度範囲において実現されるために、光照射する温度条件は、特に制限なく使用できる。例えば、１〜６０℃、１〜４０℃、４〜４０℃、２０〜４０℃の温度とすることができる。本発明は、従来は光開裂の効率が低かった低温での光照射によっても、十分に高い効率となるまで光開裂を促進できる点で、特に優れている。 Since the promotion of photocleavage according to the present invention is realized in a wide temperature range, the temperature condition for light irradiation can be used without any particular limitation. For example, it can be set as the temperature of 1-60 degreeC, 1-40 degreeC, 4-40 degreeC, 20-40 degreeC. The present invention is particularly excellent in that the photocleavage can be accelerated to a sufficiently high efficiency even by light irradiation at a low temperature where the photocleavage efficiency has been low.
本発明は、光反応による架橋開裂反応を促進しているために、光照射の際に使用される溶液の種類、塩濃度、ｐＨ等には、特に制限はない。例えば、中性付近で、例えば、ｐＨ６．８〜ｐＨ７．８の範囲で、例えば公知の緩衝液を使用して、行うことができる。 In the present invention, since the cross-linking cleavage reaction by photoreaction is promoted, there is no particular limitation on the type of solution, salt concentration, pH, etc. used in the light irradiation. For example, in the vicinity of neutrality, for example, in the range of pH 6.8 to pH 7.8, for example, using a known buffer solution.
［本発明の優位性］
本発明の優位性は、例えば、次の表１のようにまとめることができる。
[Advantages of the present invention]
The superiority of the present invention can be summarized as shown in Table 1 below, for example.
本発明による光開裂の促進は、光架橋核酸二重鎖に対して、上述の関係を満たす挿入核酸の添加によって、実現される。そのため、多様な塩基配列の光架橋核酸二重鎖が存在している場合にも、挿入核酸の塩基配列によって選択的に、特定の塩基配列の光架橋核酸二重鎖の光開裂が促進される。加熱や短波長照射による光開裂の促進は、一律の促進であるために、このような塩基配列選択性を有しない。本発明による光開裂の促進は、多様な塩基配列の光架橋核酸二重鎖の混合物から配列選択的な核酸回収ができる等の点で、特に優れている。本発明は、挿入核酸を使用した、光架橋核酸二重鎖の光開裂の配列選択的促進方法にもある。 The promotion of photocleavage according to the present invention is realized by adding an inserted nucleic acid that satisfies the above-mentioned relationship to the photocrosslinked nucleic acid duplex. Therefore, even when photocrosslinked nucleic acid duplexes of various base sequences are present, photocleavage of the photocrosslinked nucleic acid duplex of a specific base sequence is selectively promoted by the base sequence of the inserted nucleic acid. . Since the promotion of photocleavage by heating or short wavelength irradiation is uniform promotion, it does not have such base sequence selectivity. The promotion of photocleavage according to the present invention is particularly excellent in that sequence-selective nucleic acid recovery can be performed from a mixture of photocrosslinked nucleic acid duplexes having various base sequences. The present invention also resides in a method for promoting sequence selective photocleavage of a photocrosslinked nucleic acid duplex using an inserted nucleic acid.
以下に実施例をあげて、本発明を詳細に説明する。本発明は、以下に例示する実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples. The present invention is not limited to the examples illustrated below.
［光架橋核酸二重鎖の光架橋の光開裂］
［核酸鎖の調製］
次の表２に示す配列を有する核酸の鎖を調製した。
[Photocleavage of photocrosslinking of photocrosslinked nucleic acid duplex]
[Preparation of nucleic acid chain]
Nucleic acid strands having the sequences shown in Table 2 below were prepared.
表中、光応答性修飾核酸（ODN-temp）は、配列中に、次の式で表されるｃｎｖＫ（３−シアノビニルカルバゾール−１’−β−デオキシリボシド）を、光応答性修飾塩基を有するヌクレオシドとして、リン酸ジエステル結合して有している。
表中、被光架橋性核酸（ODN-pass）は、光応答性修飾核酸に対して相補的な配列を有しており、末端に標識部位として、蛍光色素（Ｃｙ３）を有している。光応答性修飾核酸は被光架橋性核酸よりも配列長が長いために、光応答性修飾核酸の鎖と被光架橋性核酸の鎖とによって形成される二重鎖は、末端に一重鎖部分(sticky end)を有する。この二重鎖に光照射すると、光応答性修飾核酸の配列中のｃｎｖＫの位置に対して相補的な位置にある被光架橋性核酸の配列中のＧに、被光架橋性核酸の配列中の３’側に隣接して位置するＴと、ｃｎｖＫとが、光架橋を形成する。図１はこの光架橋形成を示す説明図である。 In the table, the photocrosslinkable nucleic acid (ODN-pass) has a sequence complementary to the photoresponsive modified nucleic acid, and has a fluorescent dye (Cy3) as a labeling site at the end. Since the photoresponsive modified nucleic acid has a longer sequence length than the photocrosslinkable nucleic acid, the duplex formed by the photoresponsive crosslinkable nucleic acid strand and the photocrosslinkable nucleic acid strand has a single-stranded portion at the end. (sticky end). When this double strand is irradiated with light, G in the sequence of the photocrosslinkable nucleic acid at a position complementary to the position of cnvK in the sequence of the photoresponsive modified nucleic acid is added to G in the sequence of the photocrosslinkable nucleic acid. T located adjacent to the 3 ′ side of γ and cnvK form a photocrosslink. FIG. 1 is an explanatory view showing this photocrosslinking formation.
表中、挿入核酸（ODN-inv）(invader)は、光応答性修飾核酸に対して相補的な配列を有し、上記末端の一重鎖部分を有する鎖に相補的な配列をも有する。 In the table, the inserted nucleic acid (ODN-inv) (invader) has a sequence complementary to the light-responsive modified nucleic acid, and also has a sequence complementary to the strand having the single-stranded portion at the end.
［光照射による光開裂］
光応答性修飾核酸（ODN-temp）と被光架橋性核酸（ODN-pass）との二重鎖に光照射して光架橋された光架橋核酸二重鎖（dsDNA）を調製した。この光架橋10 μM dsDNA、10 μM挿入核酸（ODN-inv）（Invader strand）と100 mM NaClが50 mMカコジル酸ナトリウム(pH7.4)に溶けた溶液を37℃で1時間インキュベートした後、37℃で３６６ｎｍの光照射を行った。この光開裂のスキームを図２に示す。サンプリングしたサンプルを変性剤8M Urea ホルムアミド溶液で10倍希釈したものを変性PAGEにより解析した。サンプル 2 μMと6x loading buffer 1 μMを混合し、ゲルにアプライした。変性PAGEには8M Urea入りの15%アクリルアミドゲルを使用し、150 Vで60分間電気泳動を行った後、Cy3蛍光でのゲルのイメージングを行った。ゲル結果から、架橋体のバンドは泳動度が小さく、開裂体のバンドは泳動度が大きく出るため、バンド強度から開裂率の定量を行った。この結果を、図３及び図４に示す。Ｉｎｖ（＋）は挿入核酸（ODN-inv）を添加して実験の結果を示し、Ｉｎｖ（−）は挿入核酸（ODN-inv）を添加しない対照実験の結果を示す。
[Photocleavage by light irradiation]
A photocrosslinked nucleic acid duplex (dsDNA) was prepared by photoirradiating a double strand of a photoresponsive modified nucleic acid (ODN-temp) and a photocrosslinkable nucleic acid (ODN-pass) with light. After incubating this photocrosslinked 10 μM dsDNA, 10 μM inserted nucleic acid (ODN-inv) (Invader strand) and 100 mM NaCl in 50 mM sodium cacodylate (pH 7.4) at 37 ° C. for 1 hour, 37 Irradiation with light at 366 nm was performed at 0 ° C. The photocleavage scheme is shown in FIG. Samples that were diluted 10-fold with a denaturant 8M Urea formamide solution were analyzed by denaturing PAGE. 2 μM sample and 1 μM 6x loading buffer were mixed and applied to the gel. For denaturing PAGE, 15% acrylamide gel containing 8M Urea was used, electrophoresis was performed at 150 V for 60 minutes, and then the gel was imaged with Cy3 fluorescence. From the gel results, the band of the cross-linked product has a low migration degree and the band of the cleaved body has a high migration degree. Therefore, the cleavage rate was quantified from the band intensity. The results are shown in FIG. 3 and FIG. Inv (+) shows the result of the experiment with the addition of the inserted nucleic acid (ODN-inv), and Inv (−) shows the result of the control experiment without the addition of the inserted nucleic acid (ODN-inv).
このように、挿入核酸（ODN-inv）を添加すると、３７℃において、3時間の光照射で60%以上の開裂反応が進行していた。挿入核酸（ODN-inv）を添加しない場合には３０％程度の開裂で頭打ちになっていた。この結果から、挿入核酸による光開裂の促進は、動物や細胞の生存可能な温度範囲内で、十分に発揮されることがわかった。 Thus, when the inserted nucleic acid (ODN-inv) was added, the cleavage reaction proceeded by 60% or more at 37 ° C. by light irradiation for 3 hours. When the inserted nucleic acid (ODN-inv) was not added, it reached a peak at about 30% cleavage. From this result, it was found that the promotion of photocleavage by the inserted nucleic acid is sufficiently exerted within the temperature range in which animals and cells can survive.
［光開裂の配列選択性］
［核酸鎖の調製］
次の表３に示す配列を有する核酸の鎖を調製した。
[Sequence selectivity of photocleavage]
[Preparation of nucleic acid chain]
Nucleic acid strands having the sequences shown in Table 3 below were prepared.
挿入核酸（ODN-inv）は、光応答性修飾核酸（ODN-temp）に対して、光架橋二重鎖の分子中で二重鎖となっている塩基配列の領域においても、末端の一重鎖となる塩基配列の領域においても、いずれも相補的な塩基配列となっている。ODN-inv_rev_1は、光応答性修飾核酸（ODN-temp）に対して、光架橋二重鎖の分子中で二重鎖となっている塩基配列の領域においては相補的でないが、末端の一重鎖となる塩基配列の領域においては、相補的な塩基配列となっている。ODN-inv_rev_2は、光応答性修飾核酸（ODN-temp）に対して、光架橋二重鎖の分子中で二重鎖となっている塩基配列の領域においては相補的な塩基配列となっているが、末端の一重鎖となる塩基配列の領域においては、相補的ではない。ODN-inv_rev_3は、光応答性修飾核酸（ODN-temp）に対して、光架橋二重鎖の分子中で二重鎖となっている塩基配列の領域においても相補的ではなく、末端の一重鎖となる塩基配列の領域においても、相補的ではない。これらを用いて、架橋開裂反応の確認を行った。 The inserted nucleic acid (ODN-inv) is a single-stranded terminal in the region of the base sequence that is double-stranded in the photo-crosslinking double-stranded molecule compared to the photoresponsive modified nucleic acid (ODN-temp). Also in the region of the base sequence to be, all are complementary base sequences. ODN-inv_rev_1 is not complementary to the photo-responsive modified nucleic acid (ODN-temp) in the region of the base sequence that is double-stranded in the photo-crosslinking double-stranded molecule, but the terminal single-stranded chain In the region of the base sequence to be a complementary base sequence. ODN-inv_rev_2 is complementary to the photoresponsive modified nucleic acid (ODN-temp) in the region of the base sequence that is double-stranded in the photocrosslinking double-stranded molecule. However, it is not complementary in the region of the base sequence that becomes a single strand at the end. ODN-inv_rev_3 is not complementary to the photo-responsive modified nucleic acid (ODN-temp) even in the region of the base sequence that is double-stranded in the photo-crosslinking double-stranded molecule, and is a single-stranded single end. Even in the region of the base sequence to be, it is not complementary. These were used to confirm the cross-linking cleavage reaction.
［光照射による光開裂］
光応答性修飾核酸（ODN-temp）と被光架橋性核酸（ODN-pass）との二重鎖に光照射して光架橋された光架橋核酸二重鎖（dsDNA）を調製した。この光架橋10 μM dsDNA、10 μM Invader strandと100 mM NaClが50 mMカコジル酸ナトリウム(pH7.4)に溶けた溶液を25℃で1時間インキュベートした後、25℃で３６６ｎｍの光照射を行った。サンプリングしたサンプルを変性剤8M Urea ホルムアミド溶液で10倍希釈したものを変性PAGEにより解析した。サンプル 2 μMと6x loading buffer 1 μMを混合し、ゲルにアプライした。変性PAGEには8M Urea入りの15%アクリルアミドゲルを使用し、150 Vで60分間電気泳動を行った後、Cy3蛍光でのゲルのイメージングを行った。ゲル結果から、架橋体のバンドは泳動度が小さく、開裂体のバンドは泳動度が大きく出るため、バンド強度の開裂率の定量を行った。この結果を、図５に示す。Ｉｎｖ（＋）は挿入核酸（ODN-inv）を添加して実験の結果を示し、Ｉｎｖ（−）は挿入核酸（ODN-inv）を添加しない実験の結果を示す。図において、ODN-inv_rev_1、ODN-inv_rev_2、ODN-inv_rev_3を使用した実験の結果を、それぞれＲｅｖ１、Ｒｅｖ２、Ｒｅｖ３として示す。
[Photocleavage by light irradiation]
A photocrosslinked nucleic acid duplex (dsDNA) was prepared by photoirradiating a double strand of a photoresponsive modified nucleic acid (ODN-temp) and a photocrosslinkable nucleic acid (ODN-pass) with light. A solution of this photocrosslinked 10 μM dsDNA, 10 μM Invader strand and 100 mM NaCl in 50 mM sodium cacodylate (pH 7.4) was incubated at 25 ° C. for 1 hour, and then irradiated with light at 366 nm at 25 ° C. . Samples that were diluted 10-fold with a denaturant 8M Urea formamide solution were analyzed by denaturing PAGE. 2 μM sample and 1 μM 6x loading buffer were mixed and applied to the gel. For denaturing PAGE, 15% acrylamide gel containing 8M Urea was used, electrophoresis was performed at 150 V for 60 minutes, and then the gel was imaged with Cy3 fluorescence. From the gel results, the band of the cross-linked product has a low migration degree and the band of the cleaved body has a high migration degree. Therefore, the cleavage rate of the band intensity was quantified. The result is shown in FIG. Inv (+) shows the result of the experiment with the addition of the inserted nucleic acid (ODN-inv), and Inv (−) shows the result of the experiment without the addition of the inserted nucleic acid (ODN-inv). In the figure, the results of experiments using ODN-inv_rev_1, ODN-inv_rev_2, and ODN-inv_rev_3 are shown as Rev1, Rev2, and Rev3, respectively.
このように、挿入核酸（ODN-inv）を添加すると、２５℃において、3時間の光照射で60%以上の開裂反応が進行していた。一方、光応答性修飾核酸（ODN-temp）との相補性が不完全な塩基配列（ODN-inv_rev_1、ODN-inv_rev_2、ODN-inv_rev_3）を添加した場合には、挿入核酸（ODN-inv）を添加しない場合と同様に、光架橋の開裂反応は進行していなかった。この結果から、挿入核酸による光開裂の促進は、高い配列選択性を有していることがわかった。 Thus, when the inserted nucleic acid (ODN-inv) was added, the cleavage reaction proceeded by 60% or more at 25 ° C. by irradiation with light for 3 hours. On the other hand, when the base sequence (ODN-inv_rev_1, ODN-inv_rev_2, ODN-inv_rev_3) that is incompletely complementary to the photoresponsive modified nucleic acid (ODN-temp) is added, the inserted nucleic acid (ODN-inv) As in the case of no addition, the photocrosslinking cleavage reaction did not proceed. From this result, it was found that the promotion of photocleavage by the inserted nucleic acid has high sequence selectivity.
［光開裂の波長依存性］
同様に、光架橋体20 μM dsDNA、20 μM Invader strandと100 mM NaClが50 mMカコジル酸ナトリウム(pH7.4)に溶けた溶液を25℃で1時間インキュベートした後、25℃で、３２０ｎｍ、３３０ｎｍ、３４０ｎｍの各波長の光照射を一時間行った。サンプリングしたサンプルを変性剤8M Urea ホルムアミド溶液で10倍希釈したものを変性PAGEにより解析した。サンプル 2 μMと6x loading buffer 1 μMを混合し、ゲルにアプライした。変性PAGEには8M Urea入りの15%アクリルアミドゲルを使用し、150 Vで60分間電気泳動を行った後、Cy3蛍光でのゲルのイメージングを行った。ゲル結果から、架橋体のバンドは泳動度が小さく、開裂体のバンドは泳動度が大きく出るため、バンド強度の開裂率の定量を行った。この結果を図６に示す。
[Wavelength dependence of photocleavage]
Similarly, a solution of 20 μM dsDNA, 20 μM Invader strand and 100 mM NaCl dissolved in 50 mM sodium cacodylate (pH 7.4) was incubated at 25 ° C. for 1 hour, and then at 25 ° C., 320 nm and 330 nm. Light irradiation at each wavelength of 340 nm was performed for 1 hour. Samples that were diluted 10-fold with a denaturant 8M Urea formamide solution were analyzed by denaturing PAGE. 2 μM sample and 1 μM 6x loading buffer were mixed and applied to the gel. For denaturing PAGE, 15% acrylamide gel containing 8M Urea was used, electrophoresis was performed at 150 V for 60 minutes, and then the gel was imaged with Cy3 fluorescence. From the gel results, the band of the cross-linked product has a low migration degree and the band of the cleaved body has a high migration degree. Therefore, the cleavage rate of the band intensity was quantified. The result is shown in FIG.
このように、挿入核酸（ODN-inv）を添加すると、３２０ｎｍ、３３０ｎｍ、３４０ｎｍのいずれの波長でも、光開裂が促進されることがわかった。この結果から、挿入核酸による光開裂の促進は、広い波長範囲にわたることがわかった。 Thus, it was found that when the inserted nucleic acid (ODN-inv) was added, photocleavage was promoted at any wavelength of 320 nm, 330 nm, and 340 nm. From this result, it was found that the promotion of photocleavage by the inserted nucleic acid extends over a wide wavelength range.
本発明は、従来よりも低温で、従来よりも長波長の光照射によっても、光開裂を高い効率で進行させる技術を提供する。本発明は産業上有用な発明である。 The present invention provides a technique for allowing photocleavage to proceed with high efficiency even when irradiated with light having a longer wavelength than that of the prior art at a temperature lower than that of the prior art. The present invention is industrially useful.
Claims (13)
光架橋核酸二重鎖は、光架橋性のビニル構造を有する光応答性塩基を有する光応答性修飾核酸の鎖と、該光応答性修飾核酸に対して相補的な配列を有する被光架橋性核酸の鎖とによって二重鎖形成され、
光応答性修飾核酸の鎖と被光架橋性核酸の鎖とは、光応答性塩基によって光架橋され、
光架橋核酸二重鎖は、二重鎖の末端に一重鎖部分を有しており、
該末端の一重鎖部分を有する鎖の一重鎖部分と二重鎖部分に対して相補的な配列を有する挿入核酸の鎖を、光架橋核酸二重鎖と溶液中で接触可能に添加して、光照射する工程、
を含む、方法であり、
光架橋性のビニル構造を有する光応答性塩基を有する光応答性修飾核酸が、次の式（Ｉ）：
Ｒ１２及びＲ１３は、それぞれ独立に、シアノ基、アミド基、カルボキシル基、Ｃ２〜Ｃ７のアルコキシカルボニル基、又は水素であり、
Ｒ４は、水素を表し、
Ｒ５は、水酸基を表し、
Ｒ６は、水素又は水酸基である）
で表される修飾ヌクレオシドが、光架橋性のビニル構造を有する光応答性塩基として、リン酸ジエステル結合によって塩基配列中に導入されてなる修飾核酸である、方法。 A method of photoirradiating a photocrosslinked nucleic acid duplex by photoirradiating a photocrosslinked nucleic acid duplex having a photocrosslink formed by a photoresponsive base between strands in a nucleic acid duplex,
The photocrosslinkable nucleic acid duplex is a photocrosslinkable nucleic acid having a photoresponsive base having a photocrosslinkable vinyl structure and a sequence complementary to the photoresponsive base. Double-stranded with the nucleic acid strand,
The strand of the photoresponsive modified nucleic acid and the photocrosslinkable nucleic acid strand are photocrosslinked by a photoresponsive base,
The photocrosslinking nucleic acid duplex has a single-stranded portion at the end of the duplex,
A strand of an inserted nucleic acid having a sequence complementary to the single-stranded portion and double-stranded portion of the strand having the single-stranded portion of the terminal is added in contact with the photocrosslinking nucleic acid duplex in solution; A light irradiation step,
Including, and
A photoresponsive modified nucleic acid having a photoresponsive base having a photocrosslinkable vinyl structure is represented by the following formula (I):
R12 and R13 are each independently a cyano group, an amide group, a carboxyl group, a C2-C7 alkoxycarbonyl group, or hydrogen;
R4 represents hydrogen,
R5 represents a hydroxyl group,
R6 is hydrogen or a hydroxyl group)
The modified nucleoside represented by the above is a modified nucleic acid introduced into a base sequence by a phosphodiester bond as a photoresponsive base having a photocrosslinkable vinyl structure .
光応答性修飾核酸の塩基配列中の光架橋性のビニル構造を有する光応答性塩基と、
被光架橋性核酸の塩基配列中のＴ又はＵとの間に形成された光架橋である、請求項１に記載の方法。 Photocrosslinking formed by the light responsive bases,
A photoresponsive base having a photocrosslinkable vinyl structure in the base sequence of the photoresponsive modified nucleic acid;
The method according to claim 1, which is a photocrosslinking formed between T and U in the base sequence of the photocrosslinkable nucleic acid.
該末端の一重鎖部分が、光応答性修飾核酸の鎖の３’末端又は５’末端、または挿入核酸の鎖の３’末端又は５’末端である、請求項１〜２のいずれかに記載の方法。 The strand having the terminal single-stranded portion is a strand of a light-responsive modified nucleic acid or a strand of an inserted nucleic acid;
The single-stranded part of the terminal is the 3 'end or 5' end of the strand of the light-responsive modified nucleic acid, or the 3 'end or 5' end of the strand of the inserted nucleic acid. the method of.
末端の一重鎖部分が、光応答性修飾核酸の鎖の３’末端又は５’末端であり、
挿入核酸の鎖が、光応答性修飾核酸に対して相補的な配列を有する一重鎖である、請求項１〜５のいずれかに記載の方法。 The strand having the terminal single-stranded portion is a strand of a light-responsive modified nucleic acid,
The single-stranded part of the terminal is the 3 ′ end or 5 ′ end of the strand of the light-responsive modified nucleic acid,
The method according to any one of claims 1 to 5, wherein the strand of the inserted nucleic acid is a single strand having a sequence complementary to the light-responsive modified nucleic acid.
核酸二重鎖内の鎖間の光架橋を光開裂させて、光架橋されていない光応答性修飾核酸を、製造する方法。 By the method according to claim 1,
A method for producing a photoresponsive modified nucleic acid that is not photocrosslinked by photocleaving photocrosslinks between strands in a nucleic acid duplex.
核酸二重鎖内の鎖間の光架橋を光開裂させて、光架橋されていない被光架橋性核酸を、製造する方法。 By the method according to claim 1,
A method for producing a photocrosslinkable nucleic acid that is not photocrosslinked by photocleaving photocrosslinks between strands in a nucleic acid duplex.
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