JP7373834B2 - How the invasion complex is formed - Google Patents
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- JP7373834B2 JP7373834B2 JP2019132460A JP2019132460A JP7373834B2 JP 7373834 B2 JP7373834 B2 JP 7373834B2 JP 2019132460 A JP2019132460 A JP 2019132460A JP 2019132460 A JP2019132460 A JP 2019132460A JP 7373834 B2 JP7373834 B2 JP 7373834B2
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Description
本発明は、インベージョン複合体の形成方法等に関する。 The present invention relates to a method for forming an invasion complex, etc.
ペプチド核酸（Peptide Nucleic Acid, PNA）は1991年にNielsenらにより報告された人工核酸である。DNAの糖-リン酸骨格の代わりにN-(2-aminoethyl) glycine骨格をもつ。さらに構造に着目すると、PNAとDNAはともに単位骨格が6原子からなり、主鎖から核酸塩基までの原子数も等しい。この類似した構造のため、PNAは相補的なDNAとWatson Crick塩基対を介した二本鎖を形成する。リン酸基を有するDNAの二本鎖は、負電荷の静電反発によって不安定化するが、PNAは静電的に中性な骨格を持つため、DNAとより安定な二本鎖を形成する。PNAはDNAとの二本鎖は安定であるが、同時にDNAと同等、あるいはそれ以上の配列識別能を持ち、1塩基のミスマッチも認識が可能である。また、PNAはDNAやペプチドとは異なる非天然骨格からなるため、ヌクレアーゼやプロテアーゼなどの酵素分解に対して高い耐性を示し、遺伝子発現制御など生体内への応用が期待されている。 Peptide Nucleic Acid (PNA) is an artificial nucleic acid reported by Nielsen et al. in 1991. It has an N-(2-aminoethyl) glycine skeleton instead of the sugar-phosphate skeleton of DNA. Looking further at their structures, both PNA and DNA have unit skeletons of six atoms, and the number of atoms from the main chain to the nucleobases is the same. Because of this similar structure, PNA forms duplexes with complementary DNA through Watson Crick base pairing. DNA duplexes with phosphate groups are destabilized by electrostatic repulsion due to negative charges, but PNA has an electrostatically neutral backbone, so it forms more stable duplexes with DNA. . Although PNA is stable as a double strand with DNA, it also has a sequence recognition ability that is equal to or better than DNA, and can recognize even a single base mismatch. Furthermore, since PNA consists of a non-natural skeleton that is different from DNA and peptides, it exhibits high resistance to enzymatic degradation such as nucleases and proteases, and is expected to have in vivo applications such as controlling gene expression.
PNAの特に注目すべき性質としてインベージョンがある。インベージョンとはPNAが二本鎖のDNAに部分的に侵入する現象である。一般的に、二本鎖DNAを配列特異的に認識するには、DNAを熱などにより一本鎖に変性させ、相補的なDNAや人工核酸によって再び二本鎖を形成させる必要がある。しかし、PNAのインベージョンでは全体構造を維持したまま配列特異的にDNAを認識することが可能である。インベージョンにはいくつかの様式が報告されており、代表的なものの1つとして、double-duplex invasionが挙げられる。double-duplex invasionは互いに相補的な配列を持つ一組のPNAと、標的となるDNA二本鎖により形成されるインベージョン複合体である。配列に比較的制限がないため、応用が期待されている。 A particularly noteworthy property of PNA is invasion. Invasion is a phenomenon in which PNA partially invades double-stranded DNA. Generally, in order to recognize double-stranded DNA in a sequence-specific manner, it is necessary to denature the DNA into single-stranded DNA using heat or the like, and then form double-stranded DNA again using complementary DNA or artificial nucleic acids. However, PNA invasion allows sequence-specific DNA recognition while maintaining the overall structure. Several types of invasion have been reported, and one of the representative ones is double-duplex invasion. Double-duplex invasion is an invasion complex formed by a pair of PNAs with mutually complementary sequences and a target DNA duplex. Since there are relatively no restrictions on the arrangement, it is expected to find many applications.
天然核酸塩基で構成されたPNAを用いる場合、PNA同士の二本鎖の熱安定性はDNA/PNA二本鎖に比べて大きいため、PNA同士の二本鎖が主として生じてしまい、従来は特殊な修飾なしでインベージョンを達成することはできなかった。そこで、PNA同士の二本鎖形成を防ぐために、pseudo-complementary PNA （pcPNA）が用いられてきた（非特許文献１）。しかし、現在のpcPNAは天然核酸塩基のA、Tをそれぞれ化学修飾した2,6-ジアミノプリン（D）、2-チオウラシル（Us）を用いるものであるため、効率的な配列認識にはある程度のA、Tの割合が必要とされる。そして、一般的なペプチド合成はFmoc固相合成法が用いられるが、pcPNAの主な合成法はBoc固相合成法が用いられる（D、UsのモノマーはBoc試薬が一般的なため）。Boc法ではFmoc法よりも厳しい条件で反応を進行させなければならないため、ペプチド合成機の使用が制限されていた。 When using PNA composed of natural nucleobases, the thermal stability of double strands between PNAs is greater than that of DNA/PNA double strands, so double strands between PNAs mainly occur, and conventionally, special Invasion could not be achieved without additional qualifications. Therefore, in order to prevent double strand formation between PNAs, pseudo-complementary PNAs (pcPNAs) have been used (Non-Patent Document 1). However, current pcPNA uses 2,6-diaminopurine (D) and 2-thiouracil (U s ), which are chemically modified A and T of natural nucleobases, respectively, so there is some difficulty in efficient sequence recognition. A, T ratio of is required. The Fmoc solid phase synthesis method is generally used for peptide synthesis, but the Boc solid phase synthesis method is used as the main synthesis method for pcPNA (because Boc reagents are commonly used as monomers for D and U s ). The Boc method requires the reaction to proceed under stricter conditions than the Fmoc method, which limits the use of peptide synthesizers.
本発明は、インベージョン複合体の新規な形成方法を提供することを課題とする。より好ましくは、修飾塩基を使用する必要が無い、インベージョン複合体の形成方法を提供することを課題とする。 An object of the present invention is to provide a novel method for forming an invasion complex. More preferably, it is an object of the present invention to provide a method for forming an invasion complex that does not require the use of modified bases.
本発明者は鋭意研究を重ねた結果、2本の1本鎖人工核酸であって、（a）各々が互いに相補的な塩基配列を有し、（b）各々、モノマー間がアミド結合で連結されてなり、且つ（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させることにより、インベージョン複合体を形成できることを見出した。本発明者は、この知見に基づいてさらに研究を重ねた結果、本発明を完成させた。 As a result of extensive research, the present inventor has discovered that two single-stranded artificial nucleic acids (a) each have a mutually complementary base sequence, and (b) each monomer is linked by an amide bond. and (c) at least one of the single-stranded artificial nucleic acids contains a modification that reduces the T m value between the two single-stranded artificial nucleic acids. It was discovered that an invasion complex can be formed by contacting a double-stranded nucleic acid containing a complementary base sequence to a double-stranded nucleic acid. The present inventor completed the present invention as a result of further research based on this knowledge.
即ち、本発明は、下記の態様を包含する。 That is, the present invention includes the following aspects.
項１． 2本の1本鎖人工核酸であって、
（a）各々が互いに相補的な塩基配列を有し、
（b）各々、モノマー間がアミド結合で連結されてなり、且つ
（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、
2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む、インベージョン複合体の形成方法。
Item 1. Two single-stranded artificial nucleic acids,
(a) Each has a mutually complementary base sequence,
(b) each monomer is connected by an amide bond, and (c) at least one single-stranded artificial nucleic acid includes a modification that reduces the T m value between the two,
A method for forming an invasion complex, comprising the step of contacting two single-stranded artificial nucleic acids with a double-stranded nucleic acid containing a complementary base sequence to the two single-stranded artificial nucleic acids.
項２． 前記2本の1本鎖人工核酸がパラレル型である、及び／又は前記2本の1本鎖人工核酸を相補関係に基づいて並べた場合に対向塩基が無い塩基が存在する、項１に記載の形成方法。 Item 2. Item 1, wherein the two single-stranded artificial nucleic acids are of a parallel type, and/or when the two single-stranded artificial nucleic acids are arranged based on a complementary relationship, there is a base that has no opposing base. How to form.
項３． 前記2本の1本鎖人工核酸がPNA（Peptide Nucleic Acid）である、項１又は２に記載の形成方法。 Item 3. Item 3. The formation method according to Item 1 or 2, wherein the two single-stranded artificial nucleic acids are PNA (Peptide Nucleic Acid).
項４． 項１～３のいずれかに記載の形成方法で得られるインベージョン複合体。 Item 4. Item 3. An invasion complex obtained by the formation method according to any one of Items 1 to 3.
項５． 2本の1本鎖人工核酸であって、
（a）各々が互いに相補的な塩基配列を有し、
（b）各々、モノマー間がアミド結合で連結されてなり、且つ
（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、
2本の1本鎖人工核酸。
Item 5. Two single-stranded artificial nucleic acids,
(a) Each has a mutually complementary base sequence,
(b) each monomer is connected by an amide bond, and (c) at least one single-stranded artificial nucleic acid includes a modification that reduces the T m value between the two,
Two single-stranded artificial nucleic acids.
項６． 項５に記載の2本の1本鎖人工核酸を含む組成物。 Item 6. Item 5. A composition comprising two single-stranded artificial nucleic acids according to item 5.
項７． インベージョン複合体形成用である、項６に記載の組成物。 Section 7. Item 7. The composition according to item 6, which is for forming an invasion complex.
本発明によれば、インベージョン複合体の新規な形成方法を提供することができる。本発明の一態様においては、インベージョン法に利用する人工核酸において修飾塩基を使用する必要が無いので、該人工核酸の塩基配列設計の自由度をより高くすることや、該人工核酸をより簡便に合成することが可能であり、このような人工核酸を用いることにより、簡便にインベージョン複合体を形成することができる。 According to the present invention, a novel method for forming an invasion complex can be provided. In one aspect of the present invention, there is no need to use modified bases in the artificial nucleic acid used in the invasion method, so it is possible to increase the degree of freedom in designing the base sequence of the artificial nucleic acid, and to make the artificial nucleic acid more flexible. It can be easily synthesized, and by using such an artificial nucleic acid, an invasion complex can be easily formed.
本明細書中に示される上限及び下限は、任意に組み合わせることができる。 The upper and lower limits given herein can be combined arbitrarily.
本明細書中において、「含有」及び「含む」なる表現については、「含有」、「含む」、「実質的にからなる」及び「のみからなる」という概念を含む。 In this specification, the expressions "contain" and "including" include the concepts of "containing", "comprising", "consisting essentially" and "consisting only".
本明細書において、核酸を構成する塩基には、RNA、DNA等の天然核酸中の典型的な塩基（アデニン（A）、チミン（T）、ウラシル（U）、グアニン（G）、シトシン（C）等）のみならず、これ以外の塩基、例えばヒポキサンチン（I）、修飾塩基等も包含される。修飾塩基としては、例えば、シュードウラシル、3-メチルウラシル、ジヒドロウラシル、5-アルキルシトシン（例えば、5-メチルシトシン）、5-アルキルウラシル（例えば、5-エチルウラシル）、5-ハロウラシル（5-ブロモウラシル）、6-アザピリミジン、6-アルキルピリミジン（6-メチルウラシル）、2-チオウラシル、4-チオウラシル、4-アセチルシトシン、5-（カルボキシヒドロキシメチル）ウラシル、5’-カルボキシメチルアミノメチル-2-チオウラシル、5-カルボキシメチルアミノメチルウラシル、1-メチルアデニン、1-メチルヒポキサンチン、2，2-ジメチルグアニン、3-メチルシトシン、2-メチルアデニン、2-メチルグアニン、N6-メチルアデニン、7-メチルグアニン、5-メトキシアミノメチル-2-チオウラシル、5-メチルアミノメチルウラシル、5-メチルカルボニルメチルウラシル、5-メチルオキシウラシル、5-メチル-2-チオウラシル、2-メチルチオ-N6-イソペンテニルアデニン、ウラシル-5-オキシ酢酸、2-チオシトシン、プリン、2，6-ジアミノプリン、2-アミノプリン、イソグアニン、インドール、イミダゾール、キサンチン等が挙げられる。これらの核酸塩基は、さらに1以上の置換基を有してもよい。 In this specification, bases constituting nucleic acids include typical bases in natural nucleic acids such as RNA and DNA (adenine (A), thymine (T), uracil (U), guanine (G), cytosine (C ), etc.), but also bases other than these, such as hypoxanthine (I), modified bases, etc. Examples of the modified base include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (5- Bromouracil), 6-azapyrimidine, 6-alkylpyrimidine (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl- 2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine, N6-methyladenine, 7-Methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6-iso Examples include pentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, xanthine, and the like. These nucleobases may further have one or more substituents.
1．インベージョン複合体の形成方法
本発明は、その一態様において、2本の1本鎖人工核酸であって、（a）各々が互いに相補的な塩基配列を有し、（b）各々、モノマー間がアミド結合で連結されてなり、且つ（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む、インベージョン複合体の形成方法（本明細書において、「本発明の形成方法」と示すこともある。）に関する。以下に、これについて説明する。
1. Method for Forming Invasion Complex In one aspect, the present invention provides two single-stranded artificial nucleic acids, (a) each having a mutually complementary base sequence, (b) each having a monomer and (c) at least one of the single-stranded artificial nucleic acids contains a modification that reduces the T m value between the two. A method for forming an invasion complex (herein referred to as "formation method of the present invention") comprising a step of contacting a double-stranded nucleic acid containing a complementary base sequence to a single-stranded artificial nucleic acid of ). This will be explained below.
2本の1本鎖人工核酸（本明細書において、それぞれを、便宜的に、「1本鎖人工核酸A」、「1本鎖人工核酸B」と示すこともある。）は、（a）各々が互いに相補的な塩基配列を有する。相補的とは、塩基同士が相補関係（例えばAとT、AとU、GとC等）にあることを示す。すなわち、1本鎖人工核酸Aは1本鎖人工核酸Bの塩基配列に対して相補的な塩基配列を含み、また1本鎖人工核酸Bは1本鎖人工核酸Aの塩基配列に対して相補的な塩基配列を含む。「相補的な塩基配列」は、通常、連続した塩基配列を示すが、数箇所（例えば1箇所、2箇所、3箇所等）の断続部位を挟んでなる塩基配列も包含する。 Two single-stranded artificial nucleic acids (in this specification, for convenience, each may be referred to as "single-stranded artificial nucleic acid A" and "single-stranded artificial nucleic acid B") are (a) Each has a mutually complementary base sequence. Complementary means that the bases are complementary to each other (for example, A and T, A and U, G and C, etc.). In other words, single-stranded artificial nucleic acid A contains a base sequence that is complementary to the base sequence of single-stranded artificial nucleic acid B, and single-stranded artificial nucleic acid B contains a base sequence that is complementary to the base sequence of single-stranded artificial nucleic acid A. Contains a unique base sequence. A "complementary base sequence" usually refers to a continuous base sequence, but also includes a base sequence with several discontinuation sites (for example, one, two, three, etc.) in between.
2本の1本鎖人工核酸それぞれの塩基数は、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り特に制限されないが、例えば5塩基以上、好ましくは8塩基以上、より好ましくは10塩基以上である。特に、該塩基数を12塩基以上に設計することにより、インベージョン複合体の形成効率を大きく向上させることができる。該塩基数の上限は、例えば40塩基、好ましくは35塩基、より好ましくは30塩基、さらに好ましくは25塩基、よりさらに好ましくは20塩基である。 The number of bases in each of the two single-stranded artificial nucleic acids is not particularly limited as long as it is possible to form an invasion complex by double-duplex invasion, but for example, 5 or more bases, preferably 8 or more bases, more Preferably it is 10 bases or more. In particular, by designing the number of bases to be 12 or more bases, the efficiency of invasion complex formation can be greatly improved. The upper limit of the number of bases is, for example, 40 bases, preferably 35 bases, more preferably 30 bases, still more preferably 25 bases, even more preferably 20 bases.
1本鎖人工核酸が有する「相補的な塩基配列」の塩基数は、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り特に制限されないが、例えば1本鎖人工核酸Aと1本鎖人工核酸Bそれぞれの全塩基数100％に対して、例えば80％以上、好ましくは85％以上、より好ましくは90％以上、さらに好ましくは95％以上である。該塩基数は、全塩基数にも依るが、例えば5塩基以上、8塩基以上、10塩基以上、12塩基以上である。この場合、該塩基数の上限は、例えば40塩基、35塩基、30塩基、25塩基、20塩基である。 The number of bases in the "complementary base sequence" that a single-stranded artificial nucleic acid has is not particularly limited as long as it is possible to form an invasion complex by double-duplex invasion, but for example, single-stranded artificial nucleic acid A and single-stranded artificial nucleic acid B, for example, 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more. The number of bases depends on the total number of bases, but is, for example, 5 or more bases, 8 or more bases, 10 or more bases, or 12 or more bases. In this case, the upper limit of the number of bases is, for example, 40 bases, 35 bases, 30 bases, 25 bases, or 20 bases.
1本鎖人工核酸が有する「相補的な塩基配列」以外の塩基数は、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り特に制限されないが、例えば1本鎖人工核酸Aと1本鎖人工核酸Bそれぞれの全塩基数100％に対して、例えば20％以下、好ましくは15％以下、より好ましくは10％以下、さらに好ましくは5％以下である。また、該塩基数は、全塩基数にも依るが、例えば10塩基以下、5塩基以下、4塩基以下、3塩基以下、2塩基以下、1塩基以下である。 The number of bases other than the "complementary base sequence" that a single-stranded artificial nucleic acid has is not particularly limited as long as it is possible to form an invasion complex by double-duplex invasion. With respect to 100% of the total number of bases in each of A and single-stranded artificial nucleic acid B, the number is, for example, 20% or less, preferably 15% or less, more preferably 10% or less, and still more preferably 5% or less. Further, the number of bases depends on the total number of bases, but is, for example, 10 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, 2 bases or less, or 1 base or less.
2本の1本鎖人工核酸それぞれは、Fmoc法での合成が簡便である、モノマー合成の合成ステップが少ない、市販されていて安価である等観点から、その構成塩基として、Fmoc固相合成法で簡便にオリゴマー合成できない塩基を含まないことが好ましい。このような塩基としては、例えば2,6-ジアミノプリン、2-チオウラシル等が挙げられる。また、同様の観点から、2本の1本鎖人工核酸それぞれにおいては、その構成塩基数100％に対する典型塩基（アデニン、チミン、ウラシル、グアニン、シトシン）数の割合が高い方が好ましく、該割合は、例えば50％以上、好ましくは60％以上、より好ましくは70％以上、さらに好ましくは80％以上、よりさらに好ましくは90％以上、とりわけさらに好ましくは95％以上、特に好ましくは100％である。 Each of the two single-stranded artificial nucleic acids is synthesized using the Fmoc solid-phase synthesis method as its constituent base because it is easy to synthesize using the Fmoc method, there are few synthesis steps for monomer synthesis, and it is commercially available and inexpensive. It is preferable that it does not contain bases that cannot be easily synthesized into oligomers. Examples of such bases include 2,6-diaminopurine and 2-thiouracil. In addition, from the same viewpoint, it is preferable that the number of typical bases (adenine, thymine, uracil, guanine, cytosine) is high relative to 100% of the number of constituent bases in each of the two single-stranded artificial nucleic acids; is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, especially still more preferably 95% or more, particularly preferably 100%. .
2本の1本鎖人工核酸は、（b）各々、モノマー間がアミド結合で連結されてなる1本鎖人工核酸である。モノマーは、カルボキシ基、アミノ基、及び核酸塩基を有し、且つアミド結合で連結されることにより、他の核酸と相補的に結合可能なポリマー（人工核酸）を形成可能なものである限り、特に制限されない。モノマーには、立体異性体及び光学異性体が含まれ得るが、これらは特に限定されるものではない。モノマーとしては、例えば公知の文献（例えばChem Biol Drug Des 2017; 89: 16-37）に記載のものを採用することができる。モノマーは、例えば、窒素原子に、アミノ基を含む基、カルボキシ基を含む基、及び核酸塩基を含む基が連結してなるモノマーが挙げられる。モノマーとしてより具体的には、一般式（1）： The two single-stranded artificial nucleic acids are (b) each a single-stranded artificial nucleic acid in which monomers are linked by an amide bond. As long as the monomer has a carboxy group, an amino group, and a nucleobase, and can be linked with an amide bond to form a polymer (artificial nucleic acid) capable of complementary binding with other nucleic acids, There are no particular restrictions. Monomers may include stereoisomers and optical isomers, but are not particularly limited thereto. As the monomer, for example, those described in known literature (for example, Chem Biol Drug Des 2017; 89: 16-37) can be employed. Examples of the monomer include monomers in which a group containing an amino group, a group containing a carboxy group, and a group containing a nucleobase are linked to a nitrogen atom. More specifically, as a monomer, general formula (1):
で表される化合物が挙げられる。
Examples include compounds represented by:
R1、R2、R3、及びR4で示されるアルキル基には、直鎖状又は分岐鎖状（好ましくは直鎖状）のいずれのものも包含される。該アルキル基の炭素数は、特に制限されず、例えば1～4、好ましくは1～2、より好ましくは1である。該アルキル基の具体例としては、メチル基、エチル基、n－プロピル基、イソプロピル基、n－ブチル基、イソブチル基、tert－ブチル基、sec－ブチル基等が挙げられる。 The alkyl groups represented by R 1 , R 2 , R 3 and R 4 include either linear or branched (preferably linear) alkyl groups. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1 to 4, preferably 1 to 2, and more preferably 1. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, and the like.
R1、R2、R3、及びR4で示されるアミノ酸由来の基としては、特に制限されず、天然又は非天然の各種アミノ酸由来の基（例えばアミノ酸の1つの水素原子又は官能基を除いてなる基）を制限無く採用することができる。例えば、リシン、グルタミン、アラニン等のアミノ酸由来の基が挙げられる。 The amino acid-derived groups represented by R 1 , R 2 , R 3 , and R 4 are not particularly limited, and groups derived from various natural or non-natural amino acids (e.g., excluding one hydrogen atom or functional group of an amino acid) are not particularly limited. groups) can be employed without restriction. Examples include groups derived from amino acids such as lysine, glutamine, and alanine.
水溶性高分子としては、特に制限されず、例えばポリエチレングリコール等のポリアルキレングリコールを採用することができる。 The water-soluble polymer is not particularly limited, and for example, polyalkylene glycol such as polyethylene glycol can be used.
Baseで示される核酸塩基としては、核酸を構成する塩基を特に制限無く採用することができる。 As the nucleobase represented by Base, any base constituting a nucleic acid can be used without any particular restriction.
本発明においては、R1、R2、R3、及びR4は全て水素原子であることが特に好ましい。また、2本の1本鎖人工核酸がPNA（Peptide Nucleic Acid）であることが好ましい。 In the present invention, it is particularly preferred that R 1 , R 2 , R 3 and R 4 are all hydrogen atoms. Moreover, it is preferable that the two single-stranded artificial nucleic acids are PNA (Peptide Nucleic Acid).
2本の1本鎖人工核酸は、（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む。改変は、1本鎖人工核酸Aと1本鎖人工核酸Bとの間のTm値が減少するような改変であれば特に制限されない。 The two single-stranded artificial nucleic acids include (c) at least one of the single-stranded artificial nucleic acids includes a modification that reduces the T m value between the two. The modification is not particularly limited as long as it reduces the T m value between single-stranded artificial nucleic acid A and single-stranded artificial nucleic acid B.
改変（c）の1つとしては、例えば、（c1）2本の1本鎖人工核酸をパラレル型とする改変が挙げられる。PNA等、モノマー間がアミド結合で連結されてなる1本鎖人工核酸にもまた、DNA等のように方向性が存在する。該1本鎖人工核酸PNAにおいては、主鎖のアミド結合（－NH－C（＝O）－）における－NH－側がN末端側、－C（＝O）－側がC末端側とされている。例えばPNAでは、N末端側は、DNA等の5’末端側に対応し、C末端側はDNA等の3’末端に対応する。1本鎖核酸同士が相補的な結合により2本鎖核酸を形成する際、DNAの場合は、2本の1本鎖核酸が逆向きに結合する、すなわちそれぞれの１本鎖核酸の主鎖が逆向きになるように、核酸が相補的に結合する。本明細書においては、これをアンチパラレル型と示す。一方、PNA等、モノマー間がアミド結合で連結されてなる1本鎖人工核酸の場合も、同様に、2本鎖を形成する際は一般的にはアンチパラレル型であり、アンチパラレル型の方が安定である（Tm値が高い）（図１）。上記改変（c1）では、2本の1本鎖人工核酸の主鎖が、同じ向きである場合に、互いに相補的な塩基配列を有することになる（すなわち、パラレル型になる）ように、少なくとも一方の1本鎖人工核酸の塩基配列が設計される（図１）。2本の1本鎖人工核酸がパラレル型である場合の一例として、N末－GGGGAAAAAなる塩基配列からなる1本鎖人工核酸A’と、N末－CCCCTTTTTなる塩基配列からなる1本鎖人工核酸B’との組合せが挙げられ、これらを相補関係に基づいて並べると、下記のようになる。 One example of modification (c) is (c1) modification in which two single-stranded artificial nucleic acids are made into a parallel type. Single-stranded artificial nucleic acids, such as PNA, in which monomers are linked by amide bonds, also have directionality, like DNA. In the single-stranded artificial nucleic acid PNA, the -NH- side of the amide bond (-NH-C(=O)-) in the main chain is the N-terminal side, and the -C(=O)- side is the C-terminal side. . For example, in PNA, the N-terminal side corresponds to the 5' end of DNA, etc., and the C-terminal side corresponds to the 3' end of DNA, etc. When single-stranded nucleic acids form double-stranded nucleic acids through complementary binding, in the case of DNA, the two single-stranded nucleic acids bind in opposite directions, that is, the main strands of each single-stranded nucleic acid Nucleic acids bind complementary to each other in opposite directions. In this specification, this is referred to as an anti-parallel type. On the other hand, in the case of single-stranded artificial nucleic acids such as PNA, in which monomers are linked by amide bonds, when forming a double strand, it is generally an antiparallel type; is stable (high T m value) (Figure 1). In the above modification (c1), the main chains of two single-stranded artificial nucleic acids are at least The base sequence of one single-stranded artificial nucleic acid is designed (Figure 1). As an example of a case where two single-stranded artificial nucleic acids are of a parallel type, a single-stranded artificial nucleic acid A' consisting of a base sequence of N-terminus-GGGGAAAAAA, and a single-stranded artificial nucleic acid consisting of a base sequence of N-terminus-CCCCTTTTT. Combinations with B' are listed, and when these are arranged based on complementary relationships, they are as follows.
改変（c）の別の例としては、（c2）2本の1本鎖人工核酸を相補関係に基づいて並べた場合に対向塩基が無い塩基が存在するように設計することが挙げられる。相補関係に基づいて並べるとは、すなわち、2本の1本鎖人工核酸を、その間で対向する塩基同士の相補関係（例えばAとT、AとU、GとC等）に基づいて、相補塩基対（例えばAとT、AとU、GとC等）の数が最大となり且つ非相補塩基対（例えばAとG、GとU、TとC等）の数が最小（好ましくは0）となるように並べることを意味する。例えば、N末－AAAAAGGGGなる塩基配列からなる1本鎖人工核酸A”と、N末－CCCCCTTTTなる塩基配列からなる1本鎖人工核酸B”とを、相補関係に基づいて並べると、下記のようになる。 Another example of modification (c) is (c2) designing so that when two single-stranded artificial nucleic acids are arranged based on complementarity, there is a base that has no opposing base. Arranging based on complementary relationships means that two single-stranded artificial nucleic acids are arranged in a complementary manner based on the complementary relationships between opposing bases (for example, A and T, A and U, G and C, etc.). The number of base pairs (for example, A and T, A and U, G and C, etc.) is maximum and the number of non-complementary base pairs (for example, A and G, G and U, T and C, etc.) is minimum (preferably 0). ). For example, if a single-stranded artificial nucleic acid A" consisting of the base sequence N-terminus AAAAAAGGGG and a single-stranded artificial nucleic acid B" consisting of the base sequence N-terminus CCCCTTTT are arranged based on their complementary relationship, the following will be obtained. become.
対向塩基が無い塩基が存在するとは、すなわち、このように並べた場合に、2本の1本鎖人工核酸の少なくとも1方の一部の塩基の対向する位置に塩基が存在しないことを意味する。 The presence of a base with no opposing base means that, when arranged in this way, there is no base at the opposing position of at least one part of the bases of two single-stranded artificial nucleic acids. .
改変（c2）の具体的な例としては、例えば2本の1本鎖人工核酸の一方の塩基配列をN末端側又はC末端側にずらして設計することが挙げられる（一例として、図２を参照）。この場合、一方の1本鎖人工核酸のN末端（又はC末端）に対向塩基が無い塩基が存在することになり、一態様においてはさらに他方の1本鎖人工核酸のC末端（又はN末端）に対向塩基が無い塩基が存在することもある。本発明の一態様においては、好ましくは、一方の1本鎖人工核酸のN末端（又はC末端）に対向塩基が無い塩基が存在し、且つ他方の1本鎖人工核酸のC末端（又はN末端）に対向塩基が無い塩基が存在する（上記した、相補関係に基づいて並べた例を参照）。 A specific example of modification (c2) is to design two single-stranded artificial nucleic acids by shifting the base sequence of one of them to the N-terminus or C-terminus (as an example, reference). In this case, there will be a base with no opposing base at the N-terminus (or C-terminus) of one single-stranded artificial nucleic acid, and in one embodiment, there will be a base with no opposing base at the N-terminus (or N-terminus) of the other single-stranded artificial nucleic acid. ) may have a base that has no opposing base. In one aspect of the present invention, preferably a base with no opposing base exists at the N-terminus (or C-terminus) of one single-stranded artificial nucleic acid, and a base with no opposing base exists at the C-terminus (or N-terminus) of the other single-stranded artificial nucleic acid. There are bases that have no opposing base (see the above example of arranging based on complementarity).
改変（c2）の別の具体的な例としては、例えば、2本の1本鎖人工核酸の少なくとも一方における一部の塩基を水素原子又は他の基（例えば、芳香環（例えばベンゼン環）等の平面性分子構造）に置換えることが挙げられる。この場合、少なくとも一方の2本の1本鎖人工核酸の少なくとも一方の一部（N末端、C末端、又は末端以外の部分）に対向塩基が無い塩基が存在する。 Another specific example of modification (c2) is, for example, replacing some bases in at least one of the two single-stranded artificial nucleic acids with a hydrogen atom or other group (e.g., an aromatic ring (e.g., benzene ring), etc. (planar molecular structure). In this case, at least one of the two single-stranded artificial nucleic acids has a base that has no opposing base in a portion (N-terminus, C-terminus, or a portion other than the terminal).
改変（c2）において、1本鎖人工核酸における「対向塩基が無い塩基」の塩基数は、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り特に制限されないが、例えば1本鎖人工核酸の全塩基数100％に対して、例えば20％以下、好ましくは15％以下、より好ましくは10％以下、さらに好ましくは5％以下である。また、該塩基数は、全塩基数にも依るが、例えば1～10塩基、1～5塩基、1～4塩基、1～3塩基、1～2塩基、1塩基である。 In modification (c2), the number of bases with "no opposing base" in the single-stranded artificial nucleic acid is not particularly limited as long as it is possible to form an invasion complex by double-duplex invasion, but for example, 1 For example, it is 20% or less, preferably 15% or less, more preferably 10% or less, and even more preferably 5% or less, based on 100% of the total number of bases in the full-stranded artificial nucleic acid. Further, the number of bases depends on the total number of bases, but is, for example, 1 to 10 bases, 1 to 5 bases, 1 to 4 bases, 1 to 3 bases, 1 to 2 bases, or 1 base.
2本の1本鎖人工核酸それぞれには、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り、化学修飾されてなるものを包含することができる。具体的な化学修飾として、例えばアセチル化、ホルミル化、ミリストイル化、ピログルタミン酸化、アルキル化、グリコシル化、アミド化、アシル化、ヒドロキシル化、脱アミノ化、プレニル化、パルミトイル化、リン酸化、ビオチニル化、スクシンイミジル化等が挙げられる。 Each of the two single-stranded artificial nucleic acids can include chemically modified nucleic acids as long as it is possible to form an invasion complex by double-duplex invasion. Specific chemical modifications include, for example, acetylation, formylation, myristoylation, pyroglutamic oxidation, alkylation, glycosylation, amidation, acylation, hydroxylation, deamination, prenylation, palmitoylation, phosphorylation, and biotinylation. and succinimidylation.
2本の1本鎖人工核酸それぞれには、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り、他の物質が連結（例えば、末端に連結）している形態も包含することができる。他の分子としては、例えばリンカー、ペプチド、タンパク質、糖、アミノ酸、DNA、RNA、低分子有機・無機材料、コレステロール、デンドリマー、脂質、炭化水素、高分子材料等、さらにはこれらの複合分子が挙げられる。 Each of the two single-stranded artificial nucleic acids includes a form in which another substance is linked (for example, linked to the end) as long as it is possible to form an invasion complex by double-duplex invasion. can do. Examples of other molecules include linkers, peptides, proteins, sugars, amino acids, DNA, RNA, low-molecular organic and inorganic materials, cholesterol, dendrimers, lipids, hydrocarbons, polymeric materials, and complex molecules thereof. It will be done.
前記アミノ酸は特に限定されず、天然アミノ酸、非天然アミノ酸等を制限無く採用することができる。例えば、側鎖にアミノ基を有するリジン、ヒスチジン、またはアルギニンなどが挙げられる。中でも、リジン残基を設けることが好ましい。1本鎖人工核酸に結合させるアミノ酸の個数は特に限定はされない。通常は、1～10個程度とすることができ、1～5個程度とすることが好ましい。 The amino acids are not particularly limited, and natural amino acids, unnatural amino acids, and the like can be used without restriction. Examples include lysine, histidine, or arginine having an amino group in the side chain. Among these, it is preferable to provide a lysine residue. The number of amino acids to be bound to a single-stranded artificial nucleic acid is not particularly limited. Usually, the number can be about 1 to 10, and preferably about 1 to 5.
前記ペプチドとしては、例えば、2～40個、好ましくは2～30個、更に好ましくは6～25個のアミノ酸から構成されるペプチドが挙げられる。具体的には、細胞膜透過ペプチド［アルギニンが2～10個連結したペプチド（特に、オクタアルギニン（R8））、ペネトラチン等］、核局在化シグナルペプチド配列(HIV-1 Tat、SV40 T抗原等)、核外移行性シグナルペプチド(HIV-1 Rev、MAPKK等)、細胞膜融合ペプチド（gp41、バイアルフュージョンペプチド等）が挙げられる。 Examples of the peptide include peptides composed of 2 to 40 amino acids, preferably 2 to 30 amino acids, and more preferably 6 to 25 amino acids. Specifically, cell membrane penetrating peptides [peptides with 2 to 10 arginines linked together (especially octaarginine (R8)), penetratin, etc.], nuclear localization signal peptide sequences (HIV-1 Tat, SV40 T antigen, etc.) , nuclear export signal peptides (HIV-1 Rev, MAPKK, etc.), cell membrane fusion peptides (gp41, vial fusion peptides, etc.).
前記タンパク質としては、生体内に存在するタンパク質、薬理作用を有するタンパク質、分子認識作用を有するタンパク質等を使用でき、該タンパク質の一例として、エクスポーチン/インポーチン・タンパク質、フェブロネクチン、アビジン、抗体等を挙げることができる。 As the protein, a protein existing in the body, a protein having a pharmacological effect, a protein having a molecular recognition effect, etc. can be used. Examples of the protein include exportin/importin protein, febronectin, avidin, antibodies, etc. can be mentioned.
前記糖としては、例えば、グルコース、ガラクトース、グルコサミン、ガラクトサミン等の単糖、これらを任意に組み合わせたオリゴ糖又は多糖等が挙げられる。 Examples of the sugar include monosaccharides such as glucose, galactose, glucosamine, and galactosamine, and oligosaccharides or polysaccharides obtained by arbitrarily combining these.
前記低分子有機・無機材料としては、例えば、Cy3、Cy5等の蛍光物質；ビオチン；量子ドット；金微粒子等が挙げられる。 Examples of the low-molecular organic/inorganic materials include fluorescent substances such as Cy3 and Cy5; biotin; quantum dots; and fine gold particles.
前記デンドリマーとしては、例えば、ポリアミドアミンデンドリマー等が挙げられる。 Examples of the dendrimer include polyamide amine dendrimer.
上記脂質としては、例えば、炭素数6～50の脂肪酸、DOPE（1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine）等が挙げられる。 Examples of the above-mentioned lipids include fatty acids having 6 to 50 carbon atoms, DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), and the like.
前記炭化水素としては、例えば、炭素数6～50の飽和又は不飽和炭化水素、好ましくは炭素数6～30の直鎖状飽和又は不飽和炭化水素、特に好ましくは炭素数12～28の直鎖状飽和炭化水素が挙げられる。 Examples of the hydrocarbons include saturated or unsaturated hydrocarbons having 6 to 50 carbon atoms, preferably linear saturated or unsaturated hydrocarbons having 6 to 30 carbon atoms, particularly preferably linear saturated or unsaturated hydrocarbons having 12 to 28 carbon atoms. Examples include saturated hydrocarbons.
前記高分子材料としては、例えば、ポリエチレングリコール、ポリエチレンイミン等が挙げられる。 Examples of the polymeric material include polyethylene glycol, polyethyleneimine, and the like.
本発明の形成方法においては、上記した2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む。 The formation method of the present invention includes the step of contacting the two single-stranded artificial nucleic acids described above with a double-stranded nucleic acid containing a complementary base sequence to the two single-stranded artificial nucleic acids.
接触させる対象である2本鎖核酸は、DNA、RNAのみならず、これらに、次に例示するように、公知の化学修飾が施されたものも包含する。ヌクレアーゼなどの加水分解酵素による分解を防ぐために、各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（PS）、メチルホスホネート、ホスホロジチオネート等の化学修飾リン酸残基に置換することができる。また、各リボヌクレオチドの糖（リボース）の2位の水酸基を、-OR（Rは、例えばCH3（2´-O-Me）、CH2CH2OCH3（2´-O-MOE）、CH2CH2NHC(NH)NH2、CH2CONHCH3、CH2CH2CN等を示す）に置換してもよい。さらに、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の5位へのメチル基やカチオン性官能基の導入、あるいは2位のカルボニル基のチオカルボニルへの置換などが挙げられる。さらには、リン酸部分やヒドロキシル部分が、例えば、ビオチン、アミノ基、低級アルキルアミン基、アセチル基等で修飾されたものなどを挙げることができるが、これに限定されない。また、「ポリヌクレオチド」の語は、天然の核酸だけでなく、BNA（Bridged Nucleic Acid）、LNA（Locked Nucleic Acid）、PNA（Peptide Nucleic Acid）等の何れも包含する。2本鎖核酸は、好ましくはDNAである。 The double-stranded nucleic acids to be contacted include not only DNA and RNA, but also those subjected to known chemical modifications, as exemplified below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide are replaced with chemically modified phosphate residues such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. I can do it. In addition, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is replaced by -OR (R is, for example, CH3 (2´-O-Me), CH 2 CH 2 OCH 3 (2´-O-MOE), CH 2 CH 2 NHC(NH)NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, such as introducing a methyl group or cationic functional group to the 5-position of the pyrimidine base, or replacing the carbonyl group at the 2-position with thiocarbonyl. can be mentioned. Further examples include, but are not limited to, those in which the phosphoric acid moiety or hydroxyl moiety is modified with biotin, an amino group, a lower alkylamine group, an acetyl group, or the like. Furthermore, the term "polynucleotide" includes not only natural nucleic acids but also BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), and the like. The double-stranded nucleic acid is preferably DNA.
2本鎖核酸は、2本の1本鎖人工核酸に対して相補的な塩基配列を含む。すなわち、2本鎖核酸は、その一方の鎖において、一方の1本鎖人工核酸の塩基配列に対して相補的な塩基配列を含み、その他方の鎖において、他方の1本鎖人工核酸の塩基配列に対して相補的な塩基配列を含む。ここで、相補的とは、塩基同士が相補関係（例えばAとT、AとU、GとC等）にあることを示す。「相補的な塩基配列」は、通常、連続した塩基配列を示すが、数箇所（例えば1箇所、2箇所、3箇所等）の断続部位を挟んでなる塩基配列も包含する。好ましくは、「相補的な塩基配列」は、連続した塩基配列である。 A double-stranded nucleic acid contains a base sequence complementary to two single-stranded artificial nucleic acids. In other words, a double-stranded nucleic acid includes a base sequence complementary to the base sequence of one single-stranded artificial nucleic acid in one strand, and a base sequence complementary to the base sequence of the other single-stranded artificial nucleic acid in the other strand. Contains a complementary base sequence to the sequence. Here, complementary means that the bases are in a complementary relationship (for example, A and T, A and U, G and C, etc.). A "complementary base sequence" usually refers to a continuous base sequence, but also includes a base sequence with several discontinuation sites (for example, one, two, three, etc.) in between. Preferably, the "complementary base sequence" is a continuous base sequence.
2本鎖核酸が有する「相補的な塩基配列」の塩基数は、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り特に制限されないが、例えば2本の1本鎖人工核酸それぞれの全塩基数100％に対して、例えば80％以上、好ましくは85％以上、より好ましくは90％以上、さらに好ましくは95％以上、特に好ましくは100％である。 The number of bases in the "complementary base sequence" that a double-stranded nucleic acid has is not particularly limited as long as it is possible to form an invasion complex by double-duplex invasion. For example, it is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 100% of the total number of bases of each nucleic acid.
本発明の形成方法においては、2本鎖核酸に、上記した「2本の1本鎖人工核酸」以外の1本鎖人工核酸（1本鎖人工核酸X）を接触させることもできる。該1本鎖人工核酸Xの態様は、上記した「2本の1本鎖人工核酸」と同様であり、また上記した（a）、（b）、（c）の少なくとも1つを満たす1本鎖人工核酸であることができる。1本鎖人工核酸Xの数は、特に制限されず、例えば1～20、1～10、1～5である。1本鎖人工核酸間は、アミノ酸やリンカー等を介して連結されていてもよい。 In the formation method of the present invention, a double-stranded nucleic acid can also be brought into contact with a single-stranded artificial nucleic acid (single-stranded artificial nucleic acid X) other than the above-mentioned "two single-stranded artificial nucleic acids". The aspect of the single-stranded artificial nucleic acid The strand can be an artificial nucleic acid. The number of single-stranded artificial nucleic acids X is not particularly limited, and is, for example, 1 to 20, 1 to 10, or 1 to 5. Single-stranded artificial nucleic acids may be linked via an amino acid, a linker, or the like.
接触は、in vivoであっても、in vitroであってもよい。 Contacting may be in vivo or in vitro.
in vivoの場合、接触は通常、対象生物内又は対象細胞内で行われる。対象生物としては、例えば、ヒト、サル、マウス、ラット、イヌ、ネコ、ウサギ等の哺乳類動物； アフリカツメガエル等の両生類動物； ゼブラフィッシュ、メダカ、トラフグ等の魚類動物； ホヤ等の脊索動物； ショウジョウバエ、カイコ等の節足動物；等の動物： シロイヌナズナ、イネ、コムギ、タバコ等の植物： 酵母、アカパンカビ等の菌類： 大腸菌、枯草菌、藍藻等の細菌等が挙げられる。対象細胞としては、例えば上記生物の各種組織由来又は各種性質の細胞、例えば血液細胞、造血幹細胞・前駆細胞、配偶子（精子、卵子）、受精卵、線維芽細胞、上皮細胞、血管内皮細胞、神経細胞、肝細胞、ケラチン生成細胞、筋細胞、表皮細胞、内分泌細胞、ES細胞、iPS細胞、組織幹細胞、がん細胞等が挙げられる。 In vivo, the contact typically occurs within the subject organism or within the subject cells. Target organisms include, for example, mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; amphibians such as Xenopus; fish animals such as zebrafish, killifish, and tiger puffer fish; chordates such as sea squirts; Drosophila melanogaster Animals such as arthropods such as , silkworm; Plants such as Arabidopsis, rice, wheat, and tobacco; Fungi such as yeast and Neurospora; Bacteria such as Escherichia coli, Bacillus subtilis, and blue-green algae. Target cells include, for example, cells derived from various tissues of the above-mentioned organisms or cells with various properties, such as blood cells, hematopoietic stem cells/progenitor cells, gametes (sperm, eggs), fertilized eggs, fibroblasts, epithelial cells, vascular endothelial cells, Examples include nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.
in vitroの場合は、接触は、例えば、適当な緩衝剤溶液中で行うことができる。 In vitro, contacting can be carried out, for example, in a suitable buffer solution.
接触の態様及び条件は、double-duplex インベージョンによるインベージョン複合体の形成が可能である限り特に制限されない。in vitroの場合であれば、2本の1本鎖人工核酸と2本鎖核酸とを例えば混合することにより、両者を接触させることができる。また、in vivoの場合であれば、2本の1本鎖人工核酸を対象生物に投与する又は対象細胞に導入することにより、両者を接触させることが可能である。 The mode and conditions of contact are not particularly limited as long as formation of an invasion complex by double-duplex invasion is possible. In the case of in vitro, two single-stranded artificial nucleic acids and a double-stranded nucleic acid can be brought into contact by, for example, mixing them. Furthermore, in the case of in vivo, it is possible to bring them into contact by administering two single-stranded artificial nucleic acids to a target organism or introducing them into target cells.
2本鎖核酸に接触させる2本の1本鎖人工核酸の量は、特に制限されないが、例えば2本鎖核酸1モルに対して、例えば0.1モル以上、0.5モル以上、1モル以上、2モル以上、5モル以上、10モル以上である。該量の上限も特に制限されず、例えば500モル、100モル、50モルである。 The amount of two single-stranded artificial nucleic acids to be brought into contact with a double-stranded nucleic acid is not particularly limited, but is, for example, 0.1 mol or more, 0.5 mol or more, 1 mol or more, 2 mol or more per 1 mol of double-stranded nucleic acid. or more, 5 moles or more, 10 moles or more. The upper limit of the amount is also not particularly limited, and is, for example, 500 mol, 100 mol, or 50 mol.
斯かる本発明の形成方法により、インベージョン複合体が得られる。このため、本発明は、その一態様において、本発明の形成方法で得られるインベージョン複合体に関する。 By such a formation method of the present invention, an invasion complex can be obtained. Therefore, in one aspect, the present invention relates to an invasion complex obtained by the formation method of the present invention.
本発明の形成方法は、インベージョン複合体の特性を利用した各種用途に使用することができる。 The formation method of the present invention can be used in various applications utilizing the properties of invasion complexes.
例えば、インベージョン複合体の形成は相同組換えを誘起することや、インベージョン複合体の形成により生じた1本鎖を、1本鎖特異的切断物質（例えば、加水分解触媒（例えばCe(IV)/EDTA 錯体等）、各種ヌクレアーゼ等）により切断できることが知られている。そこで、インベージョン複合体の形成により、必要に応じて1本鎖特異的切断物質やドナーDNAをさらに加えることにより、ゲノム編集を行うことも可能である。また、インベージョン複合体の形成により、転写因子の接触や進行を阻害することにより、転写を阻害することも可能である。 For example, the formation of the invasion complex induces homologous recombination, and the single strand generated by the formation of the invasion complex is (IV)/EDTA complex, etc.), various nucleases, etc.). Therefore, by forming an invasion complex, it is also possible to perform genome editing by further adding a single-strand-specific cutting substance or donor DNA as necessary. It is also possible to inhibit transcription by inhibiting the contact and progression of transcription factors through the formation of invasion complexes.
2．2本の1本鎖人工核酸、組成物
本発明は、その一態様において、2本の1本鎖人工核酸であって、（a）各々が互いに相補的な塩基配列を有し、（b）各々、モノマー間がアミド結合で連結されてなり、且つ（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含む、2本の1本鎖人工核酸（本発明の核酸）、及び該2本の1本鎖人工核酸を含む組成物（本発明の組成物）に関する。
2. Two single-stranded artificial nucleic acids, a composition In one aspect, the present invention provides two single-stranded artificial nucleic acids, (a) each having a mutually complementary base sequence; b) two single-stranded artificial nucleic acids, each of which has monomers connected by an amide bond; and (c) at least one of the single-stranded artificial nucleic acids includes a modification that reduces the T m value between the two. (the nucleic acid of the present invention), and a composition comprising the two single-stranded artificial nucleic acids (the composition of the present invention).
2本の1本鎖人工核酸については、上記「1．インベージョン複合体の形成方法」における説明と同じである。 Regarding the two single-stranded artificial nucleic acids, the explanation is the same as in "1. Method for forming invasion complex" above.
本発明の組成物は、本発明の核酸を含有する限りにおいて特に制限されず、必要に応じてさらに他の成分を含んでいてもよい。他の成分としては、特に限定されるものではないが、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤等が挙げられる。本発明の組成物は、試薬、特にインベージョン複合体形成用組成物として使用することができる。 The composition of the present invention is not particularly limited as long as it contains the nucleic acid of the present invention, and may further contain other components as necessary. Other components include, but are not limited to, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, and thickeners. agents, humectants, coloring agents, fragrances, chelating agents, etc. The compositions of the present invention can be used as reagents, particularly compositions for forming invasion complexes.
本発明の組成物は、キットの形態であることもできる。キットにおいては、各主成分が別々の容器に収容されていてもよいし、必要に応じて核酸導入試薬、緩衝液等、本発明の形成方法の実施に必要な他の材料、試薬、器具等を適宜含んでいてもよい。 The composition of the invention can also be in the form of a kit. In the kit, each main component may be housed in a separate container, and other materials, reagents, instruments, etc. necessary for carrying out the formation method of the present invention, such as a nucleic acid introduction reagent and a buffer solution, are included as necessary. may be included as appropriate.
以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 The present invention will be described in detail below based on Examples, but the present invention is not limited to these Examples.
本実施例では、インベージョン複合体を形成するために用いる2本のPNAとして、（A） 認識配列をずらしたPNAを用いる方法（図２）、（B） PNAをparallel型に設計する方法（図１）の2手法を検討した。いずれの方法においても、相補的なPNA/PNA間の相互作用を低減させる点を重要視した。（A）の方法では、敢えて認識配列をずらしたPNAを用いることで、PNA 同士の相補的な領域が減少し、PNA二本鎖の形成が抑えられると共に、インベージョン複合体の安定性は維持されることによってインベージョン効率が向上すると考えられた。（B）の方法では、parallel 型の PNA/PNA 二本鎖の安定性が antiparallel 型の場合より低下することを利用し、片方の PNA を従来とは逆向きに合成し、parallel型になるように設計することで、PNA同士の二本鎖形成を抑制することを目指した。PNA/DNA二本鎖の安定性の低下は、parallel型にしてもPNA同士の二本鎖と比べて小さいため、インベージョン効率の向上が期待される。 In this example, the two PNAs used to form the invasion complex are (A) a method of using PNAs with shifted recognition sequences (Figure 2), and (B) a method of designing PNAs in a parallel type. We considered two methods (Figure 1). In both methods, emphasis was placed on reducing the interaction between complementary PNA/PNA. In method (A), by intentionally using PNA with a shifted recognition sequence, the complementary region between PNAs is reduced, the formation of PNA double strands is suppressed, and the stability of the invasion complex is reduced. It was thought that invasion efficiency would be improved by maintaining it. In method (B), one PNA is synthesized in the opposite direction to the conventional one, making use of the fact that the stability of a parallel-type PNA/PNA duplex is lower than that of an antiparallel-type, so that it becomes a parallel-type PNA. By designing this, we aimed to suppress double-strand formation between PNAs. The decrease in stability of the PNA/DNA duplex is smaller than that of the PNA duplex even in the parallel type, so it is expected that the invasion efficiency will be improved.
（1）PNAの合成
Fmoc固相合成法によるPNAの合成、精製を行った。合成したPNAを表１に示す。
(1) Synthesis of PNA
PNA was synthesized and purified using the Fmoc solid phase synthesis method. Table 1 shows the synthesized PNA.
全てのPNAは既報のFmocペプチド固相合成法に基づき（M. Komiyama. et al. Nat. Protc. 2008, 3, 655-662.）、自動合成機（Biotage、Syro I）を用いて合成を行った。TGR resin（20mg）をSyro I用のカラムに入れSyro Iにセットした。また、一連の合成に対する必要量の各モノマーのDMF溶液（Fmoc保護）を0.067Mで調製し、Syro Iにセットした。同様に、必要量の40% PiperidineのDMF溶液、0.050 MのHOBtと0.045 MのHBTUのDMF混合溶液、0.37M DIEPAのDMF溶液、10% Ac2OのDMF溶液を調製しSyro Iにセットした。その後、目的のPNAをSyro Iの自動合成メソッドに従って合成した。自動合成のメソッドが終了した後、手動でresinからの切り出しを行った。まず、Syro I用のカラムにDMFを500 μL加えて分散させたのち、resinの懸濁液を手動ペプチド合成用のカラム（Hipep、LibraTube）に移し、溶液を除く一連の操作を3回繰り返し行った。20% PiperidineのDMF溶液を250 μL加えて1分間の振盪後、一旦溶液を除去し、再度20% PiperidineのDMF溶液250 μLを加え、15分間振盪させ、Fmocの脱保護を行った。その後、溶液を除去し、DMFにより4回洗浄を行った。 All PNAs were synthesized using an automatic synthesizer (Biotage, Syro I) based on the previously reported Fmoc peptide solid-phase synthesis method (M. Komiyama. et al. Nat. Protc. 2008, 3, 655-662.). went. TGR resin (20mg) was placed in a Syro I column and set on Syro I. In addition, a DMF solution (Fmoc protected) of each monomer in the required amount for a series of syntheses was prepared at 0.067M and set in Syro I. Similarly, the required amounts of a DMF solution of 40% Piperidine, a DMF mixed solution of 0.050 M HOBt and 0.045 M HBTU, a DMF solution of 0.37 M DIEPA, and a DMF solution of 10% Ac 2 O were prepared and set in Syro I. . Thereafter, the desired PNA was synthesized according to the Syro I automatic synthesis method. After the automatic synthesis method was completed, the resin was manually cut out. First, add 500 μL of DMF to the column for Syro I and disperse it, then transfer the resin suspension to a column for manual peptide synthesis (Hipep, LibraTube), and repeat the series of operations except for the solution three times. Ta. After adding 250 μL of 20% Piperidine in DMF and shaking for 1 minute, the solution was removed, and 250 μL of 20% Piperidine in DMF was added again, and the mixture was shaken for 15 minutes to deprotect Fmoc. Thereafter, the solution was removed and washing was performed four times with DMF.
その後DCMにて3回洗浄し、脱樹脂を行った。TFA:H2O:triisopropylsilane（TIS）を195:2.5:2.5で混合した溶液を200 μL加え、室温で1時間静置してPNAをresinから切り出した。得られたPNA溶液を回収し、200 μLのTFAでresinを2回洗浄し、洗浄液も同様に回収した。回収したPNA溶液を約300 μLずつ2.0 mLチューブに分注し、約1.5 mLの冷ジエチルエーテルを加えてPNAを沈殿させた。遠心機で冷却しながら15,000 rpm、15分遠心することで沈殿を回収し、上清をデカンテーションにより除去した後に、新たにジエチルエーテルを加えて同様に遠心操作を行うことでPNAのペレットを洗浄した。PNAの沈殿は風乾後にMilli-Qに溶かした。PNA溶液は0.65 μmのPVDF膜のフィルターチューブによってろ過した後、逆相カラム（COSMOSIL 5C18-AR-II）を用いてHPLC精製を行った。溶離液にはMilli-Q（0.1% TFA溶液、溶離液A）とAN（0.1% TFA溶液、溶離液B）を用いた。 Thereafter, the resin was removed by washing three times with DCM. 200 μL of a solution containing TFA:H 2 O:triisopropylsilane (TIS) mixed at a ratio of 195:2.5:2.5 was added, and the mixture was allowed to stand at room temperature for 1 hour to cut out PNA from the resin. The resulting PNA solution was collected, and the resin was washed twice with 200 μL of TFA, and the washing solution was collected in the same manner. About 300 μL of the collected PNA solution was dispensed into 2.0 mL tubes, and about 1.5 mL of cold diethyl ether was added to precipitate PNA. Collect the precipitate by centrifuging at 15,000 rpm for 15 minutes while cooling in a centrifuge, remove the supernatant by decantation, and wash the PNA pellet by adding diethyl ether and performing the same centrifugation operation. did. The PNA precipitate was air-dried and then dissolved in Milli-Q. The PNA solution was filtered through a 0.65 μm PVDF membrane filter tube, and then purified by HPLC using a reverse phase column (COSMOSIL 5C 18 -AR-II). Milli-Q (0.1% TFA solution, eluent A) and AN (0.1% TFA solution, eluent B) were used as eluents.
得られたPNAをMALD-TOF MSで分析して、目的のPNAが合成されたことを確認した。 The obtained PNA was analyzed by MALD-TOF MS to confirm that the desired PNA was synthesized.
（2）インベージョン試験1
合成したPNAを用いて119 bpのDNAに対してインベージョン実験を行った。119 bpのDNAはpBFP-N1のT817-A935に一致する配列である。合成した2本のPNAと2本鎖DNAとを混合し、ゲルおよびマイクロチップ電気泳動を行い、electrophoretic mobility shift assay（EMSA）によってインベージョン複合体の形成効率を評価した。
(2) Invasion test 1
Invasion experiments were performed on 119 bp DNA using the synthesized PNA. The 119 bp DNA has a sequence matching T817-A935 of pBFP-N1. The two synthesized PNAs and double-stranded DNA were mixed and subjected to gel and microchip electrophoresis, and the efficiency of invasion complex formation was evaluated by electrophoretic mobility shift assay (EMSA).
インベージョン実験に用いる119 bpのDNA、は5’-TTCATCTGCACCACCGGCAAG-3’（配列番号19）と5’-TTGAAGAAGTCGTGGCGCTTCATG-3’ （配列番号20）をプライマーとして用い、pBFP-N1をテンプレートとしてPCRによって調製した。その後、FastGene Gel/PCR Extraction Kit（日本ジェネティクス）で精製を行い、UV-Visスペクトルによって濃度を決定した。 The 119 bp DNA used in the invasion experiment was generated by PCR using 5'-TTCATCTGCACCACCGGCAAG-3' (SEQ ID NO: 19) and 5'-TTGAAGAAGTCGTGGCGCTTCATG-3' (SEQ ID NO: 20) as primers and pBFP-N1 as a template. Prepared. Thereafter, it was purified using FastGene Gel/PCR Extraction Kit (Japan Genetics), and the concentration was determined by UV-Vis spectrum.
終濃度がDNA: 50 or 100 nM、HEPES（pH 7.0）: 5 mM、合成した各PNAの濃度がDNAのn当量（n: 実験ごとに示す）になるように、Milli-Qで調整し、10 μLの溶液を調製した。50 ℃のウォーターバス中で1時間静置し、インベージョン複合体を形成させた。インベージョン複合体の形成は、DNA/RNA分析用マイクロチップ電気泳動装置（島津、MCE-202 MultiNA）を用いて解析した。 The final concentrations were DNA: 50 or 100 nM, HEPES (pH 7.0): 5 mM, and the concentration of each synthesized PNA was adjusted using Milli-Q so that it was n equivalents of DNA (n: indicated for each experiment). A 10 μL solution was prepared. The mixture was left standing in a water bath at 50°C for 1 hour to form an invasion complex. The formation of the invasion complex was analyzed using a microchip electrophoresis device for DNA/RNA analysis (Shimadzu, MCE-202 MultiNA).
DNA/RNA分析用マイクロチップ電気泳動装置MCE-202 MultiNAを用いて測定し、得られたelectrophoretic mobility shift assay（EMSA）の結果とインベージョン実験の反応条件とをあわせて、図３～５に示す。図３は方法（A）のEMSAの結果を示し、図４は方法（B）のEMSAの結果を示し、図５はG・C塩基を多く持つ配列における方法（B）のEMSAの結果を示す。 The results of the electrophoretic mobility shift assay (EMSA) measured using the microchip electrophoresis device MCE-202 MultiNA for DNA/RNA analysis and the reaction conditions of the invasion experiment are shown in Figures 3 to 5. show. Figure 3 shows the EMSA results of method (A), Figure 4 shows the EMSA results of method (B), and Figure 5 shows the EMSA results of method (B) for sequences with many G and C bases. .
図３中の最上部、最下部に見られるバンドはそれぞれ電気泳動の基準となるマーカーによるものである。またレーン1には20 bpから500 bpのDNAを含んだラダー、レーン2では100 bpと120 bpとの間にDNAのバンドが観測された。レーン3、4では、認識配列が完全に相補的なPNAをDNAに対してそれぞれ5当量、10当量用いた結果を示し、レーン2と同じ位置にのみバンドが観測された。それに対して、レーン5、6では、認識配列を1つずらしたPNAを用いた。その結果、DNAに対してPNAを10当量加えたレーン6において、DNAのバンドとは異なる140 bpと160 bpとの間に新たなバンドが確認出来、DNAのバンドは強度が小さくなった。これはインベージョン複合体の形成により、二本鎖DNAの高次構造が変化し、泳動度に差が生じたためバンドシフトが起きたと考えられる。この結果から、認識配列をずらした方法（A）でインベージョンに成功した。 The bands seen at the top and bottom of FIG. 3 are based on markers that serve as standards for electrophoresis. Furthermore, a ladder containing DNA from 20 bp to 500 bp was observed in lane 1, and a DNA band between 100 bp and 120 bp was observed in lane 2. Lanes 3 and 4 show the results of using 5 equivalents and 10 equivalents of PNA whose recognition sequence is completely complementary to DNA, respectively, and a band was observed only at the same position as lane 2. On the other hand, in lanes 5 and 6, PNA with the recognition sequence shifted by one was used. As a result, in lane 6, where 10 equivalents of PNA were added to the DNA, a new band was observed between 140 bp and 160 bp, which was different from the DNA band, and the intensity of the DNA band became smaller. This is thought to be due to the formation of the invasion complex, which changes the higher-order structure of the double-stranded DNA and causes a difference in electrophoresis, resulting in a band shift. Based on this result, invasion was successful using method (A) in which the recognition sequence was shifted.
方法（A）の場合と同様に、図４中の最上部、最下部に見られるバンドはそれぞれ電気泳動の基準となるマーカーによるものである。またレーン1には20 bpから500 bpのDNAを含んだラダー、レーン2では100 bpと120 bpとの間にDNAのバンドが観測された。anti-parallel型のPNAを加えたレーン3、4、5では、レーン2と同じ位置にのみバンドが観測された。それに対して、parallel型でのPNAを加えたレーン6、7、8では、DNAのバンドとは異なる140 bpと160 bpとの間に新たにバンドが見られ、DNAのバンドは強度が小さくなった。これは方法（A）の場合と同様に、インベージョン複合体の形成により、二本鎖DNAの高次構造が変化し、泳動時間に差が生じたためバンドシフトが起きたと考えられる。この結果から、PNA同士がparallel型になるように設計する方法（B）でもまたインベージョンに成功した。 As in the case of method (A), the bands seen at the top and bottom of FIG. 4 are caused by markers that serve as standards for electrophoresis. Furthermore, a ladder containing DNA from 20 bp to 500 bp was observed in lane 1, and a DNA band between 100 bp and 120 bp was observed in lane 2. In lanes 3, 4, and 5 in which anti-parallel type PNA was added, a band was observed only at the same position as lane 2. On the other hand, in lanes 6, 7, and 8, in which parallel type PNA was added, a new band was observed between 140 bp and 160 bp, which was different from the DNA band, and the intensity of the DNA band became smaller. Ta. Similar to method (A), this is thought to be due to the formation of the invasion complex, which changes the higher-order structure of the double-stranded DNA and causes a difference in migration time, resulting in a band shift. Based on these results, invasion was also successful with method (B), in which PNAs are designed to be parallel to each other.
また、よりG・C塩基を多く含んだ配列についてもPNA同士がparallel型になるように設計する方法（B）を検討し、図５に結果を示した。図４の結果と同様に、anti-parallel型のPNAを加えレーン3、4では、レーン2と同じ位置にのみバンドが観測されるが、parallel型のPNAを加えレーン5、6では、DNAのバンドとは異なる140 bpと160 bpとの間に新たにバンドが見られた。したがって、よりG・C塩基を多く含んだ配列についても、PNA同士がparallel型になるように設計する方法（B）がインベージョンに有効であることを見出した。 We also investigated a method (B) in which PNAs are designed in parallel for sequences containing more G and C bases, and the results are shown in Figure 5. Similar to the results in Figure 4, in lanes 3 and 4 with anti-parallel type PNA, a band is observed only at the same position as lane 2, but in lanes 5 and 6 with parallel type PNA, the band is observed at the same position as lane 2. A new band was observed between 140 bp and 160 bp, which was different from the band. Therefore, we found that method (B), in which PNAs are designed in parallel, is effective for invasion even for sequences containing more G and C bases.
（3）インベージョン試験2
インベージョン試験1と同様にして試験した。EMSAの結果とインベージョン実験の反応条件とをあわせて、図６に示す。図６に示されるように、15mer及び12merのいずれのPNAを使用した場合も、PNA同士がparallel型になるように設計する方法（B）でインベージョンに成功した。
(3) Invasion test 2
The test was conducted in the same manner as Invasion Test 1. The results of EMSA and the reaction conditions of the invasion experiment are shown in FIG. 6. As shown in FIG. 6, when using either 15mer or 12mer PNAs, invasion was successful with method (B) in which the PNAs were designed to be parallel to each other.
（4）インベージョン試験3
インベージョン試験1と同様にして試験した。EMSAの結果とインベージョン実験の反応条件とをあわせて、図７～８に示す。図７～８に示されるように、方法（A）及び方法（B）共に、インベージョン試験1と異なる配列を使用しても、インベージョンに成功した。
(4) Invasion test 3
The test was conducted in the same manner as Invasion Test 1. The results of EMSA and the reaction conditions of the invasion experiment are shown in Figures 7 and 8. As shown in FIGS. 7 and 8, invasion was successful in both method (A) and method (B) even when a different sequence from invasion test 1 was used.
（5）Tm値測定
anti-parallel型及びparallel型のTm値を次のようにして測定した。終濃度がDNAおよびPNA: 1.0 μM、HEPES（pH 7.0）: 5 mM、になるように、Milli-Qで調整し150 μLの溶液を調製した。温度可変型の紫外可視吸光光度計（JASCO）を用い、95 ℃→25 ℃、25 ℃→95 ℃の範囲で溶液の温度を変化させながら、260 nmの波長における吸光度を測定した。得られた温度変化に伴う吸光度の変化からTm値を算出した。
(5) Tm value measurement
The Tm values of anti-parallel type and parallel type were measured as follows. A 150 μL solution was prepared using Milli-Q so that the final concentrations were 1.0 μM for DNA and PNA, and 5 mM for HEPES (pH 7.0). Using a variable temperature UV-visible spectrophotometer (JASCO), the absorbance at a wavelength of 260 nm was measured while changing the temperature of the solution in the ranges of 95°C → 25°C and 25°C → 95°C. The T m value was calculated from the obtained change in absorbance due to temperature change.
Tm値測定対象のDNA、PNAの組合せ、及びTm値を表２に示す。また、Tm値を図９にも示す。 Table 2 shows the combinations of DNA and PNA to be measured for T m value, and the T m value. The T m values are also shown in FIG.
図９から、PNA同士の二本鎖のTm値を表す左3つの絡むより、anti-parallel型では90 ℃を超える高いTm値を示すのに対し、同じ配列の場合でもparallel型では著しく安定性が低下した。そのためparallel型に設計することで効果的にPNA同士の自己会合を抑制できたと考えられる。また、右4つのカラムを見ると、anti-parallel型とparallel型での熱安定性の差が、DNA/PNAの二本鎖ではPNA同士の場合よりも小さいことが分かった。このため、インベージョン複合体全体としての安定性は十分に保持されていると考えられる。 From Figure 9, it can be seen that the anti-parallel type shows a high T m value of over 90 °C compared to the left three strands showing the T m value of double strands between PNAs, whereas the parallel type shows a significantly higher T m value of over 90 °C even in the case of the same sequence. Stability decreased. Therefore, it is thought that self-association between PNAs could be effectively suppressed by designing them in parallel. Furthermore, looking at the four columns on the right, it was found that the difference in thermal stability between the anti-parallel type and the parallel type was smaller in the case of DNA/PNA duplexes than in the case of PNAs. Therefore, it is considered that the stability of the invasion complex as a whole is sufficiently maintained.
この結果とインベージョン試験との結果より、方法（A）及び方法（B）のように、互いに相補的な2本のPNAの少なくとも一方に、両者間のTm値を減少させる改変が加えることにより、効率的にインベージョン複合体を形成できることが分かった。 Based on this result and the results of the invasion test, modifications are made to at least one of the two mutually complementary PNAs to reduce the T m value between them, as in method (A) and method (B). It was found that the invasion complex could be formed efficiently by this method.
Claims (5)
（a）各々が互いに相補的な塩基配列を有し、
（b）各々、モノマー間がアミド結合で連結されてなり、
（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含み、
（d）2本がパラレル型である、及び／又は2本を相補関係に基づいて並べた場合に対向塩基が無い塩基が存在し、且つ、
（e）天然核酸塩基、及び/又は2,6-ジアミノプリン（D）以外の修飾塩基で構成されている、あるいは天然核酸塩基、及び/又は2-チオウラシル（Us）以外の修飾塩基で構成されている、
2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む、インベージョン複合体の形成方法。 Two single-stranded artificial nucleic acids,
(a) Each has a mutually complementary base sequence,
(b) each monomer is connected by an amide bond,
(c) at least one single-stranded artificial nucleic acid includes a modification that reduces the Tm value between the two,
(d) The two bases are of a parallel type, and/or there is a base that has no opposing base when the two bases are arranged based on a complementary relationship, and
(e) composed of natural nucleobases and/or modified bases other than 2,6-diaminopurine (D), or composed of natural nucleobases and/or modified bases other than 2-thiouracil (Us); ing,
A method for forming an invasion complex, comprising the step of contacting two single-stranded artificial nucleic acids with a double-stranded nucleic acid containing a complementary base sequence to the two single-stranded artificial nucleic acids.
（a）各々が互いに相補的な塩基配列を有し、(a) Each has a mutually complementary base sequence,
（b）各々、モノマー間がアミド結合で連結されてなり、(b) each monomer is connected by an amide bond,
（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含み、(c) at least one single-stranded artificial nucleic acid includes a modification that reduces the Tm value between the two,
（d）2本がパラレル型である、及び／又は2本を相補関係に基づいて並べた場合に対向塩基が無い塩基が存在し、且つ、(d) The two bases are of a parallel type, and/or there is a base that has no opposing base when the two bases are arranged based on a complementary relationship, and
（e）天然核酸塩基、及び/又は2,6-ジアミノプリン（D）以外の修飾塩基で構成されている、あるいは天然核酸塩基、及び/又は2-チオウラシル（Us）以外の修飾塩基で構成されている、(e) composed of natural nucleobases and/or modified bases other than 2,6-diaminopurine (D), or composed of natural nucleobases and/or modified bases other than 2-thiouracil (Us); ing,
2本の1本鎖人工核酸を、前記2本の1本鎖人工核酸に対して相補的な塩基配列を含む2本鎖核酸と接触させる工程を含む、インベージョン複合体の製造方法。 A method for producing an invasion complex, comprising the step of contacting two single-stranded artificial nucleic acids with a double-stranded nucleic acid containing a complementary base sequence to the two single-stranded artificial nucleic acids.
前記2本の1本鎖人工核酸が、The two single-stranded artificial nucleic acids are
（a）各々が互いに相補的な塩基配列を有し、(a) Each has a mutually complementary base sequence,
（b）各々、モノマー間がアミド結合で連結されてなり、(b) each monomer is connected by an amide bond,
（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含み、(c) at least one single-stranded artificial nucleic acid includes a modification that reduces the Tm value between the two,
（d）2本がパラレル型である、及び／又は2本を相補関係に基づいて並べた場合に対向塩基が無い塩基が存在し、且つ、(d) The two bases are of a parallel type, and/or there is a base that has no opposing base when the two bases are arranged based on a complementary relationship, and
（e）天然核酸塩基、及び/又は2,6-ジアミノプリン（D）以外の修飾塩基で構成されている、あるいは天然核酸塩基、及び/又は2-チオウラシル（Us）以外の修飾塩基で構成されている、(e) composed of natural nucleobases and/or modified bases other than 2,6-diaminopurine (D), or composed of natural nucleobases and/or modified bases other than 2-thiouracil (Us); ing,
インベージョン複合体。Invasion complex.
（a）各々が互いに相補的な塩基配列を有し、
（b）各々、モノマー間がアミド結合で連結されてなり、
（c）少なくとも一方の1本鎖人工核酸が、両者間のTm値を減少させる改変を含み、
（d）2本がパラレル型である、及び／又は2本を相補関係に基づいて並べた場合に対向塩基が無い塩基が存在し、且つ、
（e）天然核酸塩基、及び/又は2,6-ジアミノプリン（D）以外の修飾塩基で構成されている、あるいは天然核酸塩基、及び/又は2-チオウラシル（Us）以外の修飾塩基で構成されている、
2本の1本鎖人工核酸を含む、インベージョン複合体形成用組成物。 Two single-stranded artificial nucleic acids,
(a) Each has a mutually complementary base sequence,
(b) each monomer is connected by an amide bond,
(c) at least one single-stranded artificial nucleic acid includes a modification that reduces the Tm value between the two,
(d) The two bases are of a parallel type, and/or there is a base that has no opposing base when the two bases are arranged based on a complementary relationship, and
(e) composed of natural nucleobases and/or modified bases other than 2,6-diaminopurine (D), or composed of natural nucleobases and/or modified bases other than 2-thiouracil (Us); ing,
A composition for forming an invasion complex containing two single-stranded artificial nucleic acids.
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Non-Patent Citations (4)
| Title |
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| INAGAKI, Masahito et al.，Chemistry Letter，2019年04月05日，Vol. 48, No. 4，pp. 341-344，DOI: 10.1246/cl.181048 |
| RAY, Arghya and NORDEN, BENGT，The FASEB Journal，2000年06月01日，Vol. 14, No. 9，pp. 1041-1060，DOI: 10.1096/fasebj.14.9.1041. |
| SHIGI, Narumi et al.，Molecules，2017年，Vol. 22, Article No. 1586，DOI: 10.3390/molecules22101586 |
| WITTUNG, Pernilla et al.，Nature，1994年04月07日，Vol.368，pp. 561-563，DOI: 10.1038/368561a0 |
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