Technical field
[0001]
　The present invention relates to a method for producing a hairpin-type positive-strand RNA molecule.
Background technology
[0002]
　As a technique for suppressing gene expression, for example, RNA interference (RNAi) is known (Non-Patent Document 1). A method using a short double-stranded RNA molecule called siRNA (small interfering RNA) is often used for suppressing gene expression by RNA interference. In addition, a gene expression suppression technique using a circular RNA molecule in which a partially double-stranded RNA is formed by intramolecular annealing has also been reported (Patent Document 1).
[0003]
　However, since siRNA has low stability in vivo and easily dissociates into single-stranded RNA, it is difficult to stably suppress gene expression. Patent Document 2 describes a hairpin-type single-strand long-chain RNA molecule in which the sense strand and antisense strand of siRNA are linked to a single strand using one or two linkers formed by using a cyclic amine derivative. It has been reported that siRNA can be stabilized. However, since this single-strand long RNA molecule cannot be efficiently synthesized by the phosphoramidite method using a general-purpose amidite such as TBDMS amidite, RNA amidite special for its synthesis (for example, Patent Documents 2 and 3) Must be used.
[0004]
　Patent Document 4 discloses a method of ligating a first nucleic acid strand and a second nucleic acid strand using an auxiliary nucleic acid as a third nucleic acid strand and T4 RNA ligase 2, but the auxiliary nucleic acid is long. It has been shown that the reaction is slower, and the auxiliary nucleic acids that provide good ligation efficiency in the method are limited.
Prior art literature
Patent documents
[0005]
Patent Document 1: US Patent Application Publication No. 2004/058886
Patent Document 2: International Publication WO2013 / 027843
Patent Document 3: International Publication WO2016 / 159374
Patent Document 4: International Publication WO2011 / 052013
Non-patent literature
[0006]
Non-Patent Document 1: Fire et al., Nature, (1998) Feb 19; 391 (6669): 806-811
Outline of the invention
Problems to be solved by the invention
[0007]
　An object of the present invention is to provide an efficient method for producing a hairpin-type positive-strand RNA molecule that suppresses the expression of the title gene.
Means to solve problems
[0008]
　As a result of diligent studies to solve the above problems, the present inventors have a hairpin-type single-stranded RNA molecule containing an expression-suppressing sequence for a target gene, and has a linker such as a non-nucleotide or nucleotide linker 2 By dividing into one single-stranded oligo RNA molecule, synthesizing it, annealing them, and ligating them, the hairpin-type single-stranded RNA molecule can be efficiently produced without the need for special RNA amidite or auxiliary nucleic acid. Further, they have found that the production efficiency of hairpin-type single-stranded RNA molecules with respect to the amount of enzyme used can be further increased by adjusting the ligation conditions, and have completed the present invention.
[0009]
　That is, the present invention includes the following.
[1] A method for producing a hairpin-type single-stranded RNA molecule that suppresses the expression of a target gene, which comprises
　an annealing step of annealing a first single-stranded oligo RNA molecule and a second single-stranded oligo RNA molecule. ,
　and a ligation step of ligating the ends and the Rnl2 family ligase 5 'end and the second single-stranded oligo RNA molecule' 3 of the first single-stranded oligo RNA molecule,
　the first one A double-stranded oligo RNA molecule comprises a first RNA portion and a second RNA portion linked via a first linker, one of the first RNA portion and the second RNA portion complementary to the other.
　The second single-stranded oligo RNA molecule is ligable to bind to, and comprises a third RNA portion and a fourth RNA portion linked via a second linker, and the third RNA portion and the third RNA portion. One of the RNA portions of 4 can be complementary to the other, and
　the first single-stranded oligo RNA molecule and the second single-stranded oligo RNA molecule are at the 5'end or 3'end. When an intermolecular duplex can be formed between complementary sequences and
　the first single-stranded oligo RNA molecule and the second single-stranded oligo RNA molecule form a duplex in the annealing step, The 3'end ribonucleotide residue of the first single-stranded oligo RNA molecule and the 5'end ribonucleotide residue of the second single-stranded oligo RNA molecule generate nicks, and the first single-stranded oligo RNA molecule also produces nicks. There is a gap of one or more ribonucleotide residues between the 5'end ribonucleotide residue of the single-stranded oligo RNA molecule and the 3'end ribonucleotide residue of the second single-stranded oligo RNA molecule. Exists and
　The sequence generated by the ligation of the first single-stranded oligo RNA molecule and the second single-stranded oligo RNA molecule is a
hairpin-type single-stranded RNA molecule containing a gene expression-suppressing sequence for the target gene . Production method.
[2] The first single-stranded oligo RNA molecule is represented by the following formula (I), and the second single-stranded oligo RNA molecule is represented by the following formula (II),
　　5'-Xs-. Lx 1 -Xa-3 '· · · formula
　　(I) 5'-Ya 1 -Ya 2 -Ya 3 -Lx 2 -Ys-3' · · · formula (II) 　formula (I) and formula (II) in , Xs, Xa, Ya 1 , Ya 2 , Ya 3 and Ys represent one or more ribonucleotide residues, and 　Lx 1 and Lx 2 represent the first and second linkers, respectively. , 　Ya 3 is complementary to Ys,

　Xa-Ya occurring ligation step 1 is complementary to the Xs, Xa-Ya
　occurring ligation step 1 -Ya 2 -Ya 3 includes a gene expression inhibitory sequences for the target gene, according to the above [1] Manufacturing method. [3] The first single-stranded oligo RNA molecule has uracil (U) or adenine (A) at the 3'end, and the second single-stranded oligo RNA molecule has uracil (U) at the 5'end. Or the production method according to the above [1] or [2], which has adenine (A). [4] The first linker and the second linker are each independently a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton, or (ii) a nucleotide linker, respectively. ] To [3]. [5] The production method according to any one of the above [1] to [4], wherein the ligase of the Rnl2 family is T4 RNA ligase 2. [6] The production method according to any one of [1] to [5] above, wherein the ligation is carried out in a reaction solution having a pH of 7.4 to 8.6. [7] The production method according to any one of [1] to [6] above, wherein the ligation is performed in a reaction solution containing 2 to 10 mM divalent metal ions.
The scope of the claims
[Claim 1]
　A method for producing a hairpin-type single-stranded RNA molecule that suppresses the expression of a target gene
　, which
　comprises an annealing step of annealing a first single-stranded oligo RNA molecule and a second single-stranded oligo RNA molecule, and the first . the 5 'end of the terminated second single-stranded oligo RNA molecule' 3 of the first single-stranded oligo RNA molecule and a ligation step of ligating the Rnl2 family ligase,
　the first single-stranded oligo The RNA molecule comprises a first RNA portion and a second RNA portion linked via a first linker, and one of the first RNA portion and the second RNA portion binds complementarily to the other. Possible,
　said second single-stranded oligo RNA molecule comprises a third RNA portion and a fourth RNA portion linked via a second linker, a third RNA portion and a fourth RNA. One of the portions can be complementary to the other, and
　the first single-stranded oligo RNA molecule and the second single-stranded oligo RNA molecule are complementary at the 5'end or 3'end. Intermolecular duplexes can be formed between sequences, and when
　the first single-stranded oligo RNA molecule and the second single-stranded oligo RNA molecule form a duplex in the annealing step, the first The 3'end ribonucleotide residue of the single-stranded oligo RNA molecule and the 5'end ribonucleotide residue of the second single-stranded oligo RNA molecule generate nicks, and the first single strand There is a gap of one or more ribonucleotide residues between the 5'end ribonucleotide residue of the oligo RNA molecule and the 3'end ribonucleotide residue of the second single-stranded oligo RNA molecule.
　The sequence generated by the ligation of the first single-stranded oligo RNA molecule and the second single-stranded oligo RNA molecule is a
hairpin-type single-stranded RNA molecule containing a gene expression-suppressing sequence for the target gene . Production method.
[Claim 2]
　It said first single-stranded oligo RNA molecule is represented by the following formula (I), the second single-stranded oligo RNA molecule is represented by the following formula
　　(II), 5'-Xs-Lx 1 - xa-3 '··· formula
　　(I) 5'-Ya 1 -Ya 2 -Ya 3 -Lx 2 -Ys-3' · · · formula (II) 　formula (I) and formula (II), Xs, Xa, Ya 1 , Ya 2 , Ya 3 and Ys represent one or more ribonucleotide residues, 　Lx 1 and Lx 2 represent the first linker and the second linker, respectively, and 　Ya 3 is complementary to the Ys, 　Xa-Ya occurring ligation step 1 is complementary to the Xs, 　Xa-Ya occurring ligation step 1

The production method according to claim 1 , wherein -Ya 2- Ya 3 contains a gene expression-suppressing sequence for the target gene
.
[Claim 3]
　The first single-strand oligo RNA molecule has uracil (U) or adenine (A) at the 3'end, and the second single-strand oligo RNA molecule has uracil (U) or adenine (A) at the 5'end. The production method according to claim 1 or 2, which has A).
[Claim 4]
　Claims 1 to 3, wherein the first linker and the second linker are independently (i) a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton, or (ii) a nucleotide linker. The manufacturing method according to any one item.
[Claim 5]
　The production method according to any one of claims 1 to 4, wherein the ligase of the Rnl2 family is T4 RNA ligase 2.
[Claim 6]
　The production method according to any one of claims 1 to 5, wherein the ligation is carried out in a reaction solution having a pH of 7.4 to 8.6.
[Claim 7]
　The production method according to any one of claims 1 to 6, wherein the ligation is carried out in a reaction solution containing 2 to 10 mM divalent metal ions.
[Claim 8]
　The production method according to any one of claims 1 to 7, wherein the first linker and the second linker are independently non-nucleotide linkers represented by the following formula (VI).
[Chemical 1]

[Claim 9]
　The production method according to any one of claims 1 to 8, wherein the target gene is a TGF-β1 gene, a GAPDH gene, a LMNA1 gene or an LMNA gene.
[Claim 10]
　The hairpin-type positive-strand RNA molecule consists of the nucleotide sequence represented by SEQ ID NO: 1, and the 24th and 25th ribonucleotide residues are linked via the first linker, and the 50th and 51st ribonucleotides are linked. The production method according to any one of claims 1 to 9, wherein the nucleotide residues are linked via a second linker.
[Claim 11]
　The first single-strand oligo RNA molecule and the second single-strand oligo RNA molecule are any one of the following (1) to (6), according to any one of claims 1 to 10. Manufacturing method.
(1) The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 7, in which the 24th and 25th ribonucleotide residues are linked via the first linker, and the 10th. Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 6 in which the 11th ribonucleotide residue is linked via a second linker
(2) 24th and 25th The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 19 in which the ribonucleotide residues of the above are linked via the first linker, and the 16th and 17th ribonucleotide residues are Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 18 linked via a second linker
(3) The 24th and 25th ribonucleotide residues are the first. The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 27 linked via the linker of the above and the 20th and 21st ribonucleotide residues are linked via the second linker. Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 26
(4) The 24th and 25th ribonucleotide residues are linked via the first linker. The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 29 and the 21st and 22nd ribonucleotide residues are linked via the second linker. Combination with a second single-stranded oligo RNA molecule consisting of the base sequence
(5) The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 31, in which the 24th and 25th ribonucleotide residues are linked via the first linker, and the 22nd. Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 30 in which the 23rd ribonucleotide residue is linked via a second linker
(6) 24th and 25th The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 33 in which the ribonucleotide residues of the above are linked via the first linker, and the 23rd and 24th ribonucleotide residues are Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 32 linked via a second linker.
[Claim 12]
　A single-strand oligo RNA molecule according to any one of (a) to (l) below.
(A) Single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 7 in which the 24th and 25th ribonucleotide residues are linked via a linker
(b) The 10th and 11th ribonucleotides Single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 6 in which the
residues are linked via a linker (c) The 24th and 25th ribonucleotide residues are linked via a linker. Single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 19
(d) Consists of the nucleotide sequence represented by SEQ ID NO: 18 in which the 16th and 17th ribonucleotide residues are linked via a linker. Single-stranded oligo RNA molecule
(e
) The 20th single-stranded oligo RNA molecule (f) consisting of the nucleotide sequence represented by SEQ ID NO: 27 in which the 24th and 25th ribonucleotide residues are linked via a linker. And the 21st ribonucleotide residue are linked via a linker. The single-stranded oligo RNA molecule
(g) consisting of the nucleotide sequence represented by SEQ ID NO: 26. The 24th and 25th ribonucleotide residues form the linker. Single-stranded oligo RNA molecule
(h) consisting of the nucleotide sequence represented by SEQ ID NO: 29 linked via a linker. The 21st and 22nd ribonucleotide residues are linked via a linker, which is represented by SEQ ID NO: 28. Single-stranded oligo RNA molecule consisting of the base sequence to be
(i) Single-stranded oligo RNA consisting of the base sequence represented by SEQ ID NO: 31 in which the 24th and 25th ribonucleotide residues are linked via a linker. molecule
(J) Single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 30 in which the 22nd and 23rd ribonucleotide residues are linked via a linker
(k) The 24th and 25th ribonucleotides Single-stranded oligo RNA molecule
(l) consisting of the nucleotide sequence represented by SEQ ID NO: 33 in which the residues are linked via a linker The 23rd and 24th ribonucleotide residues are linked via a linker. Single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 32
[Claim 13]
　A kit for producing a hairpin-type single-stranded RNA molecule for suppressing the expression of the TGF-β1 gene, which comprises the combination of any of the following single-stranded oligo RNA molecules (1) to (6).
(1) The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 7, in which the 24th and 25th ribonucleotide residues are linked via the first linker, and the 10th. Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 6 in which the 11th ribonucleotide residue is linked via a second linker
(2) 24th and 25th The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 19 in which the ribonucleotide residues of the above are linked via the first linker, and the 16th and 17th ribonucleotide residues are Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 18 linked via a second linker
(3) The 24th and 25th ribonucleotide residues are the first. The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 27 linked via the linker of the above and the 20th and 21st ribonucleotide residues are linked via the second linker. Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 26
(4) The 24th and 25th ribonucleotide residues are linked via the first linker. The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 29 and the 21st and 22nd ribonucleotide residues are linked via the second linker. Combination with a second single-stranded oligo RNA molecule consisting of the base sequence
(5) The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 31, in which the 24th and 25th ribonucleotide residues are linked via the first linker, and the 22nd. Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 30 in which the 23rd ribonucleotide residue is linked via a second linker
(6) 24th and 25th The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 33 in which the ribonucleotide residues of the above are linked via the first linker, and the 23rd and 24th ribonucleotide residues are Combination with a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 32 linked via a second linker.