Title of invention: Method for producing hairpin-type positive-strand RNA molecule
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). For suppressing gene expression by RNA interference, a method using a short double-stranded RNA molecule called siRNA (small interfering RNA) is often used. In addition, a technique for suppressing gene expression using a circular RNA molecule in which a partially double-stranded RNA molecule 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 an amino acid derivative. It has been reported that siRNA can be stabilized. As a method for producing this single-strand long RNA molecule, a solid-phase synthesis method by a phosphoramidite method is described in Patent Document 2.
[0004]
　As a method for producing a long-chain DNA molecule, a pair of two relatively short-chain oligo DNA molecules, that is, a first oligo DNA molecule having a carboxyl group at the end and a second oligo DNA having an amino group at the end Non-Patent Document 2 reports a technique for producing a molecule by dividing it into two molecules, synthesizing them, and linking them in a buffer containing a triazine-type dehydration condensing agent by amidation. There is no description.
[0005]
　On the other hand, the amidation reaction with an aqueous solvent has been reported in Non-Patent Document 3, for example, but there is no description regarding RNA molecules.
[0006]
　As a peptide coupling agent used to form an amide bond between a carboxylic acid of an oligomer containing a carboxylic acid at the 5'end and a label (for example, an amino group-containing substance such as cyanine, amino acid, or peptide) during solid phase synthesis. HATU is exemplified (Non-Patent Document 4).
Prior art literature
Patent documents
[0007]
Patent Document 1: US Patent Application Publication No. 2004/058886
Patent Document 2: International Publication WO2012 / 017919
Non-patent literature
[0008]
Non-Patent Document 1: Fire et al., Nature, 1998, Vol. 391, p. 806-811
Non-Patent Document 2: Liu et al., Tetrahedron, 2008, Vol. 64, p. 8417-8422.
Non-Patent Document 3: Badland et al., Tetrahedron Letters, 2017, Vol. 58, p. 4391-4394.
Non-Patent Document 4: Guidebook for the Synthesis OF Oligonucleotides Product Guide 2015/16, Link Technologies Ltd. , 2017, Vol. 58, p. 49-51.
Outline of the invention
Problems to be solved by the invention
[0009]
　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 a target gene.
Means to solve problems
[0010]
　The conventional method for producing a hairpin-type single-strand RNA molecule that suppresses the expression of a target gene is to extend the nucleoside one unit at a time by the phosphoramidite method, which cannot be said to be an efficient method.
[0011]
　Therefore, the present inventors have applied the synthesis technique of Non-Patent Document 2 to the synthesis of hairpin-type single-stranded RNA molecules for the purpose of developing an efficient method for producing hairpin-type single-stranded RNA molecules that suppress the expression of target genes. However, it was shown that the RNA molecule contains a large number of hydroxyl groups that cause side reactions in the amidation reaction, so that a large amount of impurities are generated and the desired product cannot be obtained in a high yield.
[0012]
　As a result of diligent studies to solve the above problems, the present inventors have used a predetermined type of dehydration condensing agent in a mixed solvent containing a buffer solution and a hydrophilic organic solvent such as dimethyl sulfoxide. We have found that a hairpin-type positive-strand RNA molecule can be efficiently produced in high yield by amidating the terminal of the positive-strand oligo RNA molecule, and have completed the present invention.
[0013]
　Organic solvents such as dimethyl sulfoxide have been used as nucleic acid denaturing agents in the literature (Nucleic Acid Research, 1990, Vol. 391, p.4953., Basic Molecular Biology, 4th Edition, Tamura et al., 2016, It is described in Tokyo Kagaku Dojin et al.), And it is known that such an organic solvent destabilizes nucleic acid annealing. Considering this, the above findings found by the present inventors were surprising.
[0014]
　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, in
　the presence of a dehydration condensing agent, in a mixed solvent containing a buffer solution and a hydrophilic organic solvent, according to the following formula (I). )
　Is reacted with the second single-stranded oligo RNA molecule represented by the following formula (II), and 5'-Xc-Lx 1　... · (I)
Lx 2 -X-Y-Ly-Yc-3 '· · · (II) [in the formula (I) and formula (II), X, Xc, Y and Yc consists ribonucleotide residues , Xc is complementary to X, Yc is complementary to Y, Ly is a non-nucleotide linker, Lx 1 is a non-nucleotide linker with an amino group, and Lx 2 is a non-nucleotide linker . It is a non-nucleotide linker having a carboxyl group, and XY contains a gene expression-suppressing sequence for the target gene.] 　The dehydration condensing agent is a triazine-type dehydration condensing agent, uronium containing an N-hydroxy nitrogen-containing aromatic ring structure. It is selected from the group consisting of a type dehydration condensing agent, a carbodiimide type dehydration condensing agent, a 2-halopyridinium type dehydration condensing agent, and a formamidinium 　type dehydration condensing agent. -Use in combination with hydroxy nitrogen-containing aromatic compounds or cyano (hydroxyimino) acetate,

　When the dehydration condensing agent is a 2-halopyridinium type dehydration condensing agent, it is used in combination with an N-hydroxy nitrogen-containing aromatic compound, and
　when the dehydration condensing agent is a formamidinium type dehydration condensing agent, N-hydroxy A
method for producing a hairpin-type single-stranded RNA molecule , which is used in combination with a nitrogen-containing aromatic compound or an N-hydrocarbon-substituted imidazole derivative .
[0015]
[2] The production method according to [1], wherein the linker Ly is a non-nucleotide linker having an amino acid skeleton or an amino alcohol skeleton, and the linker Lx 2 is a non-nucleotide linker having an amino acid skeleton.
[0016]
[3] The Ly is a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton, or a non-nucleotide linker containing -NHCH 2 COO-, and Lx 2 is a pyrrolidine skeleton and a piperidine having a carboxyl group. The production method according to [1] or [2], which is a non-nucleotide linker containing at least one of the skeletons or a non-nucleotide linker containing -NHCH 2 COOH.
[0017]
[4] Described in any of [1] to [3], wherein Lx 1 is represented by the following formula (III) and Lx 2 is represented by the following formula (IV) or the following formula (IV'). Production method.
[Chemical

formula 1] [In formula (III), R 1 is an optionally substituted alkylene chain, and -OR 1 is attached to the 3'end of Xc via a phosphodiester bond]
[Chemical formula 1] 2]

[In formula (IV), R 2 is an optionally substituted alkylene chain, p is 1 or 2, and -OR 2 is a phosphodiester bond at the 5'end of X. In formula (IV'), R 2 is an optionally substituted alkylene chain and -OR 2 is attached to the 5'end of X via a phosphodiester bond].
[0018]
[5] (i) The uronium-type dehydration condensing agent containing the N-hydroxy nitrogen-containing aromatic ring structure is a benzotriazolyluronium-type dehydration condensing agent.
　(Ii) The N-hydroxy nitrogen-containing aromatic compound is ,
　(Iii) the above cyano (hydroxyimino) acetate is a cyano (hydroxyimimino) alkyl ester, and / or
　(iv) the above N-hydrocarbon substituted imidazole derivative.
The production method according to any one of [1] to [4] , which is an N-alkylimidazole derivative .
[0019]
[6] The production method according to [1] to [5], wherein the hydrophilic organic solvent is a hydrophilic aprotic organic solvent.
[0020]
[7] The production method according to [6], wherein the hydrophilic aprotic organic solvent is dimethyl sulfoxide, N, N-dimethylformamide, N, N'-dimethylethyleneurea, or acetonitrile.
[0021]
[8] The dehydration condensing agent is 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride, O- (7-azabenzotriazole-1-). Il) -N, N, N', N'-tetramethyluronium hexafluorophosphate, N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, 2-chloro-1-methylpyridinium iodine Do, or chloro-N, N, N', N'-tetramethylformamidinium hexafluorophosphate, and the
　N-hydroxy nitrogen-containing aromatic compound is 1-hydroxy-7-azabenzotriazole.
The above-mentioned
one of [1] to [7] , wherein the cyano (hydroxyimino) acetate is ethyl cyano (hydroxyimino) acetate, and the N-hydrocarbon substituted imidazole derivative is N-methylimidazole. Production method.
[0022]
[9] The combination of the dehydration condensing agent and the hydrophilic aprotic organic solvent is O- (7-azabenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexa. Combination of fluorophosphate and dimethyl sulfoxide, O- (7-azabenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate and N, N-dimethylformamide Combination of N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole and dimethyl sulfoxide, or chloro-N, N, N', N' The production method according to any one of [7] and [8], which is a combination of tetramethylformamidinium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and dimethyl sulfoxide.
[0023]
[10] The production method according to any one of [1] to [9], wherein the pH of the buffer solution is 6.5 to 7.5.
[0024]
[11] Ly is a non-nucleotide linker containing a pyrrolidine skeleton or a piperidine skeleton, Lx 1 is a non-nucleotide linker having an amino group, and Lx 2 is a pyrrolidine skeleton and a piperidine skeleton having a carboxyl group. The production method according to any one of [1] to [10], which is a non-nucleotide linker containing at least one of the above.
[0025]
[12] The production method according to any one of [1] to [11], wherein Ly is represented by the following formula (V).
[Chemical 3]

[0026]
[13] The production method according to any one of [1] to [12], wherein Lx 1 is represented by the following formula (VI) and Lx 2 is represented by the following formula (VII).
[Chemical 4]

[Chemical 5]

[0027]
[14] The production method according to any one of [1] to [13], wherein the target gene is a TGF-β1 gene.
[0028]
[15] The production method according to any one of [1] to [14], wherein the hairpin-type single-strand RNA molecule comprises the base sequence represented by SEQ ID NO: 1.
[0029]
[16] The single-stranded oligo RNA molecule according to (a) or (b) below.
(A) 1 th ribonucleotide residues Lx 2 is connected to the 26 th and 27 th single strand consisting of the nucleotide sequence of ribonucleotide residues represented by SEQ ID NO: 3 which are connected via a Ly oligo RNA molecule
(b) 1 th ribonucleotide residues Lx 2 is connected with, made of 26 th and 27 th nucleotide sequence ribonucleotide residues represented by SEQ ID NO: 6 which is connected via a Ly Single-stranded oligo RNA molecule
[0030]
[17] A kit for producing a hairpin-type single-stranded RNA molecule for suppressing the expression of a target gene, which comprises the combination of the single-stranded oligo RNA molecules according to (1) or (2) below.
(1) The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 2 in which the 24th ribonucleotide residue is linked to Lx 1 , and the first ribonucleotide residue is Lx 2. Combination of the second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 3, in which the 26th and 27th ribonucleotide residues are linked via Ly
(2) 22nd ribonucleotide residues Lx of 1 and the first single-stranded oligo RNA molecule comprising the nucleotide sequence represented by SEQ ID NO: 5 which is connected with, first ribonucleotide residues Lx 2 is connected with, 26 A combination of a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 6, in which the 27th and 27th ribonucleotide residues are linked via Ly.
[0031]
　This specification includes the disclosure content of Japanese Patent Application No. 2018-187767, which is the basis of the priority of the present application.
Effect of the invention
[0032]
　According to the present invention, a hairpin-type single-strand RNA molecule containing an expression-suppressing sequence for a target gene can be efficiently produced.
A brief description of the drawing
[0033]
FIG. 1 is a schematic view of the manufacturing method of the present invention. The vertical lines (solid lines) above the lines representing the X, Xc, Y, and Yc sequences in the figure schematically indicate that the sequences are complementary, but the individual ribonucleotide residues and their correspondences. It does not limitly represent the complementarity with the residues to be produced. The boundary between the array X and the array Y in the figure is schematically represented by a vertical line (dotted line).
FIG. 2 is a schematic diagram of the ssTbRNA molecule (SEQ ID NO: 1). Lx 1 represents a non-nucleotide linker having an amino group, Ly represents a non-nucleotide linker, and Lx 2 represents a non-nucleotide linker having a carboxyl group. Positions 29 (U) to 47 (C) of SEQ ID NO: 1 correspond to active sequences (gene expression-suppressing sequences for the TGF-β1 gene).
Mode for carrying out the invention
[0034]
　Hereinafter, the present invention will be described in detail.
[0035]
　The present invention relates to a method for producing a hairpin-type positive-strand RNA molecule that suppresses gene expression. The hairpin-type single-stranded RNA molecule produced by the method of the present invention contains a non-nucleotide linker at the 3'end of the sense strand and the 5'end of the antisense strand of the double-stranded RNA containing a gene expression-suppressing sequence. It has a single-stranded structure in which ribonucleotides are further linked via a sequence that is linked via a sequence and contains an additional non-nucleotide linker at the 3'end of the antisense strand. The 5'end and 3'end of the hairpin-type positive-strand RNA molecule produced by the method of the present invention are not bound. As used herein, the term "hairpin type" means that a single-stranded RNA molecule forms one or more double-stranded structures by intramolecular annealing (self-annealing). The hairpin-type positive-strand RNA molecule produced by the method of the present invention has at least 2 by intramolecular annealing of the 5'side region containing the 5'end and the 3'side region containing the 3'end separately. Form two double chain structures. In the present specification, "RNA", "RNA molecule", "nucleic acid molecule" and "nucleic acid" may be composed only of nucleotides, but nucleotides and non-nucleotide substances (for example, amino acids such as proline derivatives and glycine derivatives). It may be composed of a derivative).
[0036]
　In the present invention, a hairpin-type single-stranded RNA molecule containing a gene expression-suppressing sequence is used as (i) a first single-stranded oligo RNA molecule having a non-nucleotideed linker (Lx 1 ) having an amino group on the 3'side. , (Ii) Two single-stranded oligo RNA molecules having a non-nucleotideed linker (Lx 2 ) having a carboxyl group on the 5'side and a non-nucleotideed linker (Ly) on the 3'side. It can be produced by dividing into fragments, synthesizing them, and linking them by an amidation reaction. Here, the carboxyl group is also referred to as a carboxylic acid group. A schematic diagram of the method of the present invention is shown in FIG. In FIG. 1, Lx 1 is a non-nucleotide linker having an amino group, Lx 2 is a non-nucleotide linker having a carboxyl group, and Ly is a non-nucleotide linker. In the method of the present invention, a relatively long-chain hairpin-type single-strand RNA molecule can be produced by linking a pair of shorter-chain single-strand RNA molecules, whereby high production efficiency can be realized. ..
[0037]
　More specifically, the method for producing a hairpin-type single-strand RNA molecule containing a gene expression-suppressing sequence according to the present invention is the first one represented by the following formula (I) in the presence of a dehydration condensing agent. It comprises a step of reacting a strand oligo RNA molecule with a second single strand oligo RNA molecule represented by the following formula (II).
[0038]
　5'-Xc-Lx 1　... (I)
　Lx 2 -XY-Ly-Yc-3'... (II)
[0039]
　As will be described later, when a carbodiimide type dehydration condensing agent is used as the dehydration condensing agent in the above method, the carbodiimide type dehydration condensing agent is an N-hydroxy nitrogen-containing aromatic compound (for example, hydroxybenzotriazole or a derivative thereof) or cyano ( Hydroxyimino) Used in combination with acetate ester. When a 2-halopyridinium type dehydration condensing agent is used as the dehydration condensing agent, the 2-halopyridinium type dehydration condensing agent is used in combination with an N-hydroxy nitrogen-containing aromatic compound. When a formamidinium type dehydration condensing agent is used as the dehydration condensing agent, the formamidinium type dehydration condensing agent is used in combination with an N-hydroxy nitrogen-containing aromatic compound or an N-hydrocarbon-substituted imidazole derivative.
[0040]
　The reaction between the first single-strand oligo RNA molecule and the second single-strand oligo RNA molecule is an amidation reaction. A first single-stranded oligo RNA molecule (specifically , an amino group at Lx 1 at the end of this molecule ) and a second single-stranded oligo RNA molecule (specifically, Lx at the end of this molecule). By the amidation reaction (dehydration condensation reaction) of the carboxyl group (2), an amide bond can be formed between the first single-stranded oligo RNA molecule and the second single-stranded oligo RNA molecule. This amidation reaction in the production method of the present invention can be carried out in a reaction solvent, particularly in a mixed solvent containing a buffer solution and a hydrophilic organic solvent.
[0041]
　In the present invention, the "oligoRNA" and the "oligoRNA molecule" refer to an RNA molecule having a base sequence of 49 base length or less (the number of residues of the non-nucleotide linker is not included). In the present invention, the terms "oligoRNA" and "oligoRNA molecule" are generally used interchangeably. The single-stranded oligo RNA molecule according to the present invention may also be referred to as a single-stranded oligo RNA, an oligo nucleic acid, a single-stranded nucleic acid molecule, an oligo RNA, or an oligo RNA molecule.
[0042]
　Formula (I) represents a structure in which the sequence Xc and the linker Lx 1 are linked in the order of Xc-Lx 1 from the 5'end side . Formula (II) represents a structure in which the sequences X, Y and Yc and the linkers Lx 2 and Ly are linked in the order of Lx 2 -XY-Ly-Yc from the 5'end side .
[0043]
　In formulas (I) and (II), X, Xc, Y and Yc consist of ribonucleotide residues (one or more ribonucleotide residues). The ribonucleotide residue may have any nucleobase selected from adenine, uracil, guanine or cytosine. The ribonucleotide residue may also be a modified ribonucleotide residue, and may have, for example, a modified nucleobase (modified base). Modifications include, but are not limited to, fluorescent dye labeling, methylation, halogenation, pseudouridineization, amination, deamination, thiolation, dihydrolation and the like. Each of X, Xc, Y and Yc may independently consist of only unmodified ribonucleotide residues, or may contain modified ribonucleotide residues in addition to unmodified ribonucleotide residues. It may consist only of modified ribonucleotide residues.
[0044]
　In the present invention, Xc is complementary to X. Xc preferably consists of a sequence (complementary sequence) that is completely complementary to the entire region of X or a partial region thereof. When Xc is completely complementary to the entire region of X, Xc consists of complementary sequences of the entire region of X from the 5'end to the 3'end, and the base numbers of Xc and X are the same. Further, when Xc is completely complementary to the partial region of X, Xc consists of a complementary sequence of the partial region of X, and Xc is 1 or more bases than X, for example, 1 to 4 bases or 1 to 2 It has a small number of bases. This partial region is preferably a region consisting of a base sequence continuous from the terminal base on the Lx 2 side in X.
[0045]
　X may be, for example, 19 to 39 bases long, preferably 19 to 30 bases long, and more preferably 19 to 25 bases long.
[0046]
　Xc may be, for example, 19 to 39 bases long, preferably 19 to 30 bases long, and more preferably 19 to 25 bases long.
[0047]
　In the present invention, Yc and Y have the same base length. In the present invention, Yc is complementary to Y. Yc preferably consists of a sequence that is completely complementary to the entire region of Y (complementary sequence). When Yc is completely complementary to the entire region of Y, Yc consists of complementary sequences of the entire region from the 5'end to the 3'end of Y, and the base numbers of Yc and Y are the same.
[0048]
　Y may be, for example, 1 to 5 bases long, preferably 1 to 3 bases long, and more preferably 1 or 2 bases long.
[0049]
　Yc may be, for example, 1 to 5 bases long, preferably 1 to 3 bases long, and more preferably 1 or 2 bases long.
[0050]
　The difference [(X + Y)-(Xc + Yc)] between the total number of bases (base length) of X and Y [(X + Y)] and the total number of bases (base length) of Xc and Yc [(Xc + Yc)] is Although not particularly limited, it is, for example, 0 to 4 bases (base length), preferably 0, 1, or 2 bases (base length).
[0051]
　In the second single-stranded oligo RNA molecule represented by the formula (II), the total number of bases of X, Y and Yc is, for example, 21 to 49 bases, preferably 21 to 30 bases, more preferably 25 bases. ~ 30 bases.
[0052]
　The linker Lx 1 in the above formula (I) is a non-nucleotide linker having an amino group, preferably a primary amino group or a secondary amino group. The amino group is a linker Lx 1 may be present at the end of, but may be present therein, it is preferably present at the end.
　The linker Lx 1 is represented by, for example, the following formula (III).
[0053]
[Chemical 6]

[0054]
　In formula (III), R 1 is an alkylene chain having m carbon atoms. Here, the alkylene chain R 1 optional hydrogen atoms on the carbon atoms of the may be substituted with any substituent or may be unsubstituted. m is not particularly limited and can be appropriately set according to the desired length of the linker Lx 1 , but for example, from the viewpoint of manufacturing cost and yield, m is preferably an integer of 1 to 30, and more preferably. It is an integer of 1 to 20, and more preferably an integer of 1 to 10. -OR 1 in formula (III) is attached to the 3'end of Xc via a phosphodiester bond. That is, the linker Lx 1 -OR in 1 is attached via a position to phosphodiester bonds 3 'terminus of the sugar (ribose)' Xc 3.
[0055]
　The linker Lx 1 preferably has a linear structure. In a preferred embodiment, the linker Lx 1 may be represented by the following formula (VI).
[0056]
[Chemical 7]

[0057]
　The linker Lx 2 in the above formula (II) is a non-nucleotide linker having a carboxyl group.
[0058]
　The linker Lx 2 may have an arbitrary structure as long as it has a carboxyl group and does not inhibit the amidation reaction. The carboxyl group is a linker Lx 2 may be present at the end of, but may be present therein, it is preferably present at the end.
[0059]
Examples of the 　linker Lx 2 include non-nucleotide linkers having an amino acid skeleton. Such a linker Lx 2 may be a linker derived from an amino acid and containing a structure having a carboxyl group at the terminal.
[0060]
　As used herein, the term "amino acid skeleton" refers to any organic compound containing at least one amino group and one or more carboxyl groups in the molecule, or a structure thereof. Here, the amino group refers to a functional group obtained by removing one or more hydrogen atoms from ammonia, a primary amine, or a secondary amine. The amino group and / or carboxyl group constituting the amino acid skeleton may form an arbitrary chemical bond such as an amide bond or an ester bond. The amino acid from which the amino acid skeleton is derived may be, for example, a natural amino acid or an artificial amino acid. The natural amino acid refers to an amino acid having a naturally occurring structure or an optical isomer thereof. In addition, artificial amino acids refer to amino acids with structures that do not exist in nature. That is, the artificial amino acid is an amino acid, that is, a carboxylic acid derivative containing an amino group (an organic compound containing at least one amino group and a carboxyl group in the molecule), and refers to a carboxylic acid derivative having a structure that does not exist in nature. The artificial amino acid preferably does not contain, for example, a heterocycle. Amino acids include, for example, glycine, α-alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamine, glutamate, histidine, isoleucine, leucine, lysine, hydroxylysine, methionine, phenylalanine, serine, threonine, tyrosine, valine, proline , 4-Hydroxyproline, tryptophan, β-alanine, 1-amino-2-carboxycyclopentane, aminobenzoic acid, aminopyridinecarboxylic acid, nipecotic acid, isonipecotic acid, pipecoric acid, 3-pyrrolidincarboxylic acid, γ-aminobutyric acid , Or sarcosin, and may or may not have a substituent or a protective group. Substituents include, but are not limited to, halogen, hydroxy, alkoxy, amino, carboxy, sulfo, nitro, carbamoyl, sulfamoyl, alkyl, alkenyl, alkynyl, haloalkyl, aryl, arylalkyl, alkylaryl, cycloalkyl, etc. Cycloalkoxy, cycloal Examples thereof include killalkyl, cyclylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, silyl, silyloxyalkyl, pyrrolyl, imidazolyl, and the like. The protecting group may be, for example, a functional group that inactivates a highly reactive functional group, and examples thereof include known protecting groups. For protecting groups, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. The protecting group is not particularly limited, and is, for example, tert-butyldimethylsilyl group, bis (2-acetoxyethyloxy) methyl group, triisopropylsilyloxymethyl group, 1- (2-cyanoethoxy) ethyl group, 2-cyano. Examples thereof include an ethoxymethyl group, a 2-cyanoethyl group, a trilsulfonylethoxymethyl group, a trityl group and a dimethoxytrityl group. Further, the amino acid may be any isomer if an isomer such as an optical isomer, a geometric isomer, or a three-dimensional isomer is present. These may be single isomers or mixtures.
[0061]
Preferred examples of the 　linker Lx 2 include a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton having a carboxyl group, or a non-nucleotide linker containing -NHCH 2 COOH. In one embodiment, the linker Lx 2 is represented by, for example, the following formula (IV) or the following formula (IV'), and more preferably the following formula (IV).
[0062]
[Chemical 8]

[0063]
　In formula (IV) and formula (IV'), R 2 is an alkylene chain having n carbon atoms. Here, the alkylene chain R 2 optional hydrogen atoms on the carbon atoms of the may be substituted with any substituent or may be unsubstituted. n is not particularly limited and can be appropriately set according to the desired length of the linker Lx 2. However, for example, n is preferably an integer of 1 to 30 from the viewpoint of manufacturing cost, yield, and the like, and more preferably. It is an integer of 1 to 20, and more preferably an integer of 1 to 10.
[0064]
　In formula (IV), p is 1 or 2, preferably 1.
[0065]
　-OR 2 in formula (IV) and formula (IV') is attached to the 5'end of X via a phosphodiester bond. That is, the linker Lx 2 -OR in 2 is attached through a phosphodiester linkage to the 5 'position of the terminal sugar (ribose)' X 5.
[0066]
　Here, the sum of m of R 1 and n of R 2 is, for example, an integer of 2 to 31, preferably an integer of 2 to 21, and more preferably an integer of 2 to 15.
[0067]
　In formula (IV), linker Lx 2 has a cyclic structure such as a pyrrolidine skeleton or a piperidine skeleton.
[0068]
　In a preferred embodiment, the linker Lx 2 may be represented by the following formula (VII).
[0069]
[Chemical 9]

[0070]
　The linker represented by the formula (VII) may be an optically active substance represented by the following formula (VII-1) or the following formula (VII-2).
[0071]
[Chemical 10]

[0072]
[Chemical 11]

[0073]
　In another embodiment, the linker Lx 2 may be represented by the following formula (VII').
[0074]
[Chemical 12]

[0075]
　The linker Ly in formula (II) is a non-nucleotide linker. The linker Ly may have any structure as long as it does not inhibit the amidation reaction. Examples of the linker Ly include non-nucleotide linkers having an amino acid skeleton or an amino alcohol skeleton. Such a linker Ly may be a linker containing a structure derived from an amino acid. This "amino acid skeleton" is as described above. However, the amino group and / or the carboxyl group constituting the amino acid skeleton of the linker Ly may form an arbitrary chemical bond such as an amide bond or an ester bond.
[0076]
　Here, the "amino alcohol skeleton" refers to an arbitrary organic compound containing at least one amino group and one or more hydroxyl groups in the molecule, or a structure thereof. The "amino group" is as described above. The amino group and / or the hydroxyl group constituting the amino alcohol skeleton of the linker Ly may form any chemical bond such as an amide bond, an ester bond, and an ether bond. Examples of the amino alcohol include an amino alcohol obtained by converting the carboxyl group of the above amino acid. The amino alcohol may be, for example, aminoethanol, aminopropanol, valinol, prolinol, or piperidinemethanol, and may or may not have a substituent or protecting group. Substituents include, but are not limited to, halogen, hydroxy, alkoxy, amino, carboxy, sulfo, nitro, carbamoyl, sulfamoyl, alkyl, alkenyl, alkynyl, haloalkyl, aryl, arylalkyl, alkylaryl, cycloalkyl, etc. Cycloalkenyl, cycloalkylalkyl, cyclylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, silyl, silyloxyalkyl, pyrrolyl, imidazolyl, and the like can be mentioned. The protecting group may be, for example, a functional group that inactivates a highly reactive functional group, and examples thereof include known protecting groups. For protecting groups, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. The protecting group is not particularly limited, and is, for example, tert-butyldimethylsilyl group, bis (2-acetoxyethyloxy) methyl group, triisopropylsilyloxymethyl group, 1- (2-cyanoethoxy) ethyl group, 2-cyano. Examples thereof include an ethoxymethyl group, a 2-cyanoethyl group, a trilsulfonylethoxymethyl group, a trityl group and a dimethoxytrityl group. Also, Amino Arco The isomer may be any isomer if an isomer such as an optical isomer, a geometric isomer, or a three-dimensional isomer is present. These may be single isomers or mixtures.
[0077]
　The linker Ly is preferably a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton, or a non-nucleotide linker containing -NHCH 2 COO-. It is more preferable that the linker Ly has a cyclic structure that can be substituted at two or more positions. The linker Ly may have one or more n-membered rings (eg, n = 5 or 6), and if it has two or more rings, they may have a fused ring, a spiro ring, a bicyclo ring, or the like. It may be formed. The linker Ly may have one or more substituents. The linker Ly links the above Y and Yc via any two groups thereof (for example, a substituent on two atoms constituting the n-membered ring). The hairpin-type single-strand RNA molecule produced by the method of the present invention is excellent in nuclease resistance because the sense strand and the antisense strand are linked by such a linker.
[0078]
　In one embodiment, the linker Ly is a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton, preferably a non-nucleotide linker containing a pyrrolidine skeleton or a piperidine skeleton. The pyrrolidine skeleton may be, for example, the skeleton of a pyrrolidine derivative in which one or more carbon atoms constituting the 5-membered ring of pyrrolidine are substituted, and when substituted, for example, other than the carbon atom (C-2) at the 2-position. It is preferable that the carbon atom of is substituted. The carbon atom may be replaced with, for example, a nitrogen atom, an oxygen atom or a sulfur atom. The pyrrolidine skeleton may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the 5-membered ring of pyrrolidine. In the pyrrolidine skeleton, for example, a hydrogen atom may be bonded to the carbon atom and the nitrogen atom constituting the 5-membered ring of pyrrolidine, or a substituent as described later may be bonded. The linker Ly may link Y and Yc, for example, via any group of the pyrrolidine skeleton described above. They can be linked via any one carbon atom constituting the 5-membered ring and a nitrogen atom, preferably a carbon atom (C-2) at the 2-position of the 5-membered ring and a nitrogen atom. Examples of the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton.
[0079]
　In one embodiment, in the present invention, in formulas (I) and (II), Ly is a non-nucleotide linker containing a pyrrolidine skeleton or a piperidine skeleton, and Lx 1 is a non-nucleotide having an amino group. A linker, Lx 2 is a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton having a carboxyl group.
[0080]
　The piperidine skeleton may be, for example, the skeleton of a piperidine derivative in which one or more carbon atoms constituting the 6-membered ring of piperidine are substituted, and when substituted, for example, other than the carbon atom (C-2) at the 2-position. It is preferable that the carbon atom of is substituted. The carbon atom may be replaced with, for example, a nitrogen atom, an oxygen atom or a sulfur atom. The piperidine skeleton may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the 6-membered ring of piperidine. In the above piperidine skeleton, the carbon atom and nitrogen atom constituting the 6-membered ring of piperidine may be bonded with, for example, a hydrogen atom or a substituent as described later. The linker Ly may link Y and Yc, for example, via any group of the piperidine skeletons described above. They can be linked via any one carbon atom constituting the 6-membered ring and a nitrogen atom, preferably a carbon atom (C-2) at the 2-position of the 6-membered ring and a nitrogen atom.
　The linker Ly is represented by, for example, the following formula (VIII).
[0081]
[Chemical 13]

[0082]
　In formula (VIII), X 1 and X 2 are independently H 2 , O, S or NH, and
Y 1 and Y 2 are independently single bonds, CH 2 , NH, O, respectively. Or S, where
Z is a hydrogen atom or substituent attached to C-3, C-4, C-5 or C-6 on ring A, and
L 1 is an alkylene chain having r carbon atoms. Here, the hydrogen atom on the carbon atom of the alkylene chain may or may not be substituted with OH, OR a , NH 2 , NHR a , NR a R b , SH or SR a , or ,
L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms,
where Y 1 is used.However, in the case of NH, O or S, the atom of L 1 bonded to Y 1 is carbon, the atom of L 1 bonded to O is carbon, the oxygen atoms are not adjacent to each other, and L 2 is carbon. It is an alkylene chain of several s, where the hydrogen atom on the carbon atom of the alkylene chain is substituted even if it is substituted with OH, OR a , NH 2 , NHR a , NR a R b , SH or SR a. It does not have to be, or L 2 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with oxygen atoms, where Y 2 is NH, O or S. , The atom of L 2 bonded to Y 2 is carbon, the atom of L 2 bonded to O is carbon, the oxygen atoms are not adjacent to each other, and R

a and R b are independent substituents or protective groups;
q is 1 or 2,
r is an integer in the range 0-30, and
s is in the range 0-30. It is an integer, and in
ring A, one carbon atom other than C-2 on ring A may be replaced with a nitrogen atom, an oxygen atom, or a sulfur atom, and a carbon-carbon double bond is formed in ring A. Alternatively, it may contain a carbon-nitrogen double bond.
[0083]
　Y and Yc are linked to the linker Ly via -OL 1 or -OL 2 in formula (VIII) . In one embodiment, Y may be linked to the linker Ly via -OL 1 and Yc via -OL 2 . In another embodiment, Y may be linked to the linker Ly via -OL 2 and Yc via -OL 1 .
[0084]
　In formula (VIII), X 1 and X 2 are, for example, H 2 , O, S or NH , respectively, independently of each other. In formula (VIII), the fact that X 1 is H 2 means that X 1 forms CH 2 together with the carbon atom to which X 1 is bonded . The same applies to X 2 .
[0085]
　In formula (VIII), Y 1 and Y 2 are independently single bonds, CH 2 , NH, O or S, respectively.
[0086]
　In formula (VIII), in ring A, q is 1 or 2. When q is 1, ring A is a 5-membered ring, for example, the pyrrolidine skeleton. Examples of the pyrrolidine skeleton include a proline skeleton, a prolinol skeleton, and the like, and these divalent structures can be exemplified. When q is 2, ring A is a 6-membered ring, for example, the piperidine skeleton. In ring A, one carbon atom other than C-2 on ring A may be replaced with a nitrogen atom, an oxygen atom or a sulfur atom. Further, the ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond in the ring A. Ring A may be, for example, either L-type or D-type.
[0087]
　In formula (VIII), Z is a hydrogen atom or substituent attached to C-3, C-4, C-5 or C-6 on ring A. When Z is a substituent, the substituent Z may be 1, a plurality, or may not exist, and when there are a plurality of substituents, they may be the same or different.
[0088]
　The substituent Z is, for example, halogen, OH, OR a , NH 2 , NHR a , NR a R b , SH, SR a or an oxo group (= O).
[0089]
　R a and R b are, for example, independent substituents or protecting groups, and may be the same or different. The substituents include, for example, halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cyclylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl. , Heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl and the like. The same applies hereinafter. Substituent Z may be these listed substituents.
[0090]
　The protecting group is, for example, a functional group that inactivates a highly reactive functional group, and examples thereof include known protecting groups. For the protecting group, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. The protecting group is not particularly limited, and is, for example, a tert-butyldimethylsilyl group (hereinafter, TBDMS group), a bis (2-acetoxyethyloxy) methyl group (hereinafter, ACE group), and a triisopropylsilyloxymethyl group (hereinafter, ACE group). , TOM group), 1- (2-cyanoethoxy) ethyl group (hereinafter, CEE group), 2-cyanoethoxymethyl group (hereinafter, CEM group), 2-cyanoethyl group (hereinafter, CE group), trilsulfonylethoxymethyl Examples thereof include a group (hereinafter, TEM group), a trityl group (hereinafter, Tr group) and a dimethoxytrityl group (hereinafter, DMTr group). When Z is OR a , examples of the protecting group include TBDMS group, ACE group, TOM group, CEE group, CEM group and TEM group. In addition to this, a silyl-containing group can also be mentioned. The same applies hereinafter.
[0091]
　In formula (VIII), L 1 is an alkylene chain having r carbon atoms. The hydrogen atom on the alkylene carbon atom may or may not be substituted with , for example, OH, OR a , NH 2 , NHR a , NR a R b , SH or SR a . Alternatively, L 1 may be a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with oxygen atoms. The polyether chain is, for example, polyethylene glycol. When Y 1 is NH, O or S, the atom of L 1 bonded to Y 1 is carbon, the atom of L 1 bonded to O is carbon, and the oxygen atoms are not adjacent to each other.
[0092]
　In formula (VIII), L 2 is an alkylene chain having s carbon atoms. The hydrogen atom on the carbon atom of the alkylene chain may or may not be substituted with , for example, OH, OR a , NH 2 , NHR a , NR a R b , SH or SR a . Alternatively, L 2 may be a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with oxygen atoms. When Y 2 is NH, O or S, the atom of L 2 bonded to Y 2 is carbon, the atom of L 2 bonded to O is carbon, and the oxygen atoms are not adjacent to each other.
[0093]
The r of 　L 1 and the s of L 2 are not particularly limited, and the lower limit thereof is, for example, 0, and the upper limit is not particularly limited. r and s can be appropriately set according to the desired length of the linker Ly, for example. r and s are, for example, preferably an integer of 0 to 30, more preferably an integer of 0 to 20, and even more preferably an integer of 0 to 15, independently of each other in terms of manufacturing cost, yield, and the like. is there. r and s may be the same or different.
[0094]
　R a and R b are the same as described above.
[0095]
　In formula (VIII), hydrogen atoms may be independently substituted with halogens such as Cl, Br, F and I, respectively.
[0096]
　In a preferred embodiment, the linker Ly may be represented by any of the following formulas (VIII-1) to (VIII-9). In the following formulas (VIII-1) to (VIII-9), t is an integer of 0 to 10, and r and s are the same as in the above formula (VIII). Specific examples include r = 8 in the formula (VIII-1), r = 3 in the formula (VIII-2), r = 4 or 8 in the formula (VIII-3), and r = 7 in the formula (VIII-4). Or 8, r = 3 and s = 4 in formula (VIII-5), r = 8 and s = 4 in formula (VIII-6), r = 8 and s = 4 in formula (VIII-7), formula ( In VIII-8), r = 5 and s = 4, and in the formula (VIII-9), t = 1 and s = 4.
[0097]
[Chemical 14]

[0098]
　In one embodiment, the linker Ly may be represented by the following formula (V) or the following formula (IX).
[0099]
[Chemical 15]

[0100]
[Chemical 16]

[0101]
　The linker represented by the formula (V) may be an optically active substance represented by the following formula (V-1) or the following formula (V-2).
[0102]
[Chemical 17]

[0103]
[Chemical 18]

[0104]
　In another embodiment, the linker Ly is a non-nucleotide linker containing -NHCH 2 COO-, such a linker is represented, for example, by the formula (XXI) below. This linker Ly basically corresponds to the linker Ly represented by the above formula (VIII), and the description of the linker represented by the above formula (VIII) is described in the linker represented by the following formula (XXI). Is also used.
[0105]
[Chemical 19]

[0106]
　In formula (XXI), X 1 and X 2 are independently H 2 , O, S or NH, and
Y 1 and Y 2 are independently single bonds, CH 2 , NH, O, respectively. Or S, where
L 1 is an alkylene chain having r carbon atoms, where the hydrogen atoms on the carbon atoms of the alkylene chain are OH, OR a , NH 2 , NHR a , NR a R b , SH or It may or may not be substituted with SR a , or
L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain is substituted with an oxygen atom,
where Y When 1 is NH, O or S, Y 1L bound to 1 atom is carbon and L bound to O 1 atom of carbon, between the oxygen atom is not adjacent,
L 2 is an alkylene chain with carbon number s, wherein the alkylene The hydrogen atom on the carbon atom of the chain may or may not be substituted with OH, OR a , NH 2 , NHR a , NR a R b , SH or SR a , or
L 2 is When one or more carbon atoms of the alkylene chain are polyether chains substituted with oxygen atoms,
where Y 2 is NH, O or S, the atom of L 2 bonded to Y 2 is carbon. in and, L is bonded to O 2 atoms of carbon, between the oxygen atom is not adjacent, R a and R b
Are independently substituents or protecting groups;
r is an integer in the range 0-30 and
s is an integer in the range 0-30.
[0107]
　Y and Yc are linked to the linker Ly via -OL 1 or -OL 2 in formula (XXI) . In one embodiment, Y may be linked to the linker Ly via -OL 1 and Yc via -OL 2 . In another embodiment, Y may be linked to the linker Ly via -OL 2 and Yc via -OL 1 .
[0108]
　In formula (XXI), X 1 and X 2 are, for example, H 2 , O, S or NH , respectively, independently of each other. In formula (XXI), the fact that X 1 is H 2 means that X 1 forms CH 2 together with the carbon atom to which X 1 is bonded . The same applies to X 2 .
[0109]
　In formula (XXI), Y 1 and Y 2 are independently single bonds, CH 2 , NH, O or S, respectively.
[0110]
　R a and R b are, for example, independent substituents or protecting groups, and may be the same or different. The substituents include, for example, halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cyclylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl. , Heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl and the like. The same applies hereinafter.
[0111]
　The protecting group is, for example, a functional group that inactivates a highly reactive functional group, and examples thereof include known protecting groups. For the protecting group, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. The protecting group is not particularly limited, and examples thereof include a TBDMS group, an ACE group, a TOM group, a CEE group, a CEM group, a CE group, a TEM group, a Tr group, and a DMTr group. The same applies hereinafter.
[0112]
　In formula (XXI), L 1 is an alkylene chain having r carbon atoms. The hydrogen atom on the alkylene carbon atom may or may not be substituted with , for example, OH, OR a , NH 2 , NHR a , NR a R b , SH or SR a . Alternatively, L 1 may be a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with oxygen atoms. The polyether chain is, for example, polyethylene glycol. When Y 1 is NH, O or S, the atom of L 1 bonded to Y 1 is carbon, the atom of L 1 bonded to O is carbon, and the oxygen atoms are not adjacent to each other.
[0113]
　In formula (XXI), L 2 is an alkylene chain having s carbon atoms. The hydrogen atom on the carbon atom of the alkylene chain may or may not be substituted with , for example, OH, OR a , NH 2 , NHR a , NR a R b , SH or SR a . Alternatively, L 2 may be a polyether chain in which one or more carbon atoms of the alkylene chain are replaced with oxygen atoms. When Y 2 is NH, O or S, the atom of L 2 bonded to Y 2 is carbon, the atom of L 2 bonded to O is carbon, and the oxygen atoms are not adjacent to each other.
[0114]
The r of 　L 1 and the s of L 2 are not particularly limited, and the lower limit thereof is, for example, 0, and the upper limit is not particularly limited. r and s can be appropriately set according to the desired length of the linker Ly, for example. r and s are, for example, preferably an integer of 0 to 30, more preferably an integer of 0 to 20, and even more preferably an integer of 0 to 15, independently of each other in terms of manufacturing cost, yield, and the like. is there. r and s may be the same or different.
[0115]
　R a and R b are the same as described above.
[0116]
　In formula (XXI), hydrogen atoms may be independently substituted with halogens such as Cl, Br, F and I, respectively.
[0117]
In a preferred embodiment, the linker Ly may be represented by the following formula (XXII).
[0118]
[Chemical 20]

[0119]
　The first and second single-stranded oligo RNA molecules can be prepared using RNA synthesis methods known to those skilled in the art. Examples of RNA synthesis methods known to those skilled in the art include a phosphoramidite method and an H-phosphonate method. In the phosphoramidite method, ribonucleoside bound to the hydrophobic group of the carrier is extended by a condensation reaction with RNA amidite (ribonucleoside phosphoramidite), and after oxidation and deprotection, the condensation reaction with RNA amidite is repeated. This makes it possible to perform RNA synthesis.
[0120]
　The first single-stranded oligo RNA molecule represented by the formula (I) can be prepared by a general RNA synthesis method, for example, a phosphoramidite method. In one embodiment, pre-linker Lx 1 aminoalcohol solid support is bonded through an amino group corresponding to the structure of (e.g., later-described 3'-PT Amino-Modifier C4 CPG ; Link Technologies) using a normal According to the method, it can be produced by solid-phase synthesis of Xc from the 3'end side. Any RNA amidite can be used for the synthesis, for example, various protecting groups such as TBDMS group, TOM group, ACE group, CEE group, CEM group, TEM group and DMTr group are added to the hydroxyl group at the 2'position. General-purpose RNA amidite having can also be used. Further, as the solid phase carrier, any solid phase carrier such as a polystyrene carrier, an acrylamide carrier, or a glass carrier can be used. The carrier may be in any form such as beads, plates, chips, tubes and the like. Examples of the carrier to which the amino alcohol is bound include, but are not limited to, 3'-PT Amino-Modifier C3 CPG, C7 CPG (Link Technologies) and the like.
[0121]
　Similarly, the second single-stranded oligo RNA molecule represented by the formula (II) can be prepared by a general RNA synthesis method, for example, a phosphoramidite method. In one embodiment, Yc is solid-phase synthesized on a solid-phase carrier from the 3'-terminal side as usual, and then the linker Ly is ligated to the 5'-terminal of Yc. Next, solid-phase synthesis is further performed from the end thereof, and the linker Lx 2 is linked to the 5'end of X to prepare a second single-stranded oligo RNA molecule. Any RNA amidite can be used for the synthesis, for example, various protecting groups such as TBDMS group, TOM group, ACE group, CEE group, CEM group, TEM group and DMTr group are added to the hydroxyl group at the 2'position. General-purpose RNA amidite having can also be used. Further, as the solid phase carrier, any solid phase carrier such as a polystyrene carrier, an acrylamide carrier, or a glass carrier can be used. The carrier may be in any form such as beads, plates, chips, tubes and the like. Examples of the carrier include, 　but are not limited to, NittoPhase (R) HL rG (ibu), rU (KINOVATE), and the like.
[0122]
Any suitable RNA synthesis monomer can be used for linking 　the linker Lx 2 to the 5'end of X, and for example, an RNA synthesis monomer represented by the following formula (X) can be used. it can. This monomer basically corresponds to the linker represented by the above formula (IV), and the description of the linker represented by the above formula (IV) is also incorporated into the monomer represented by the following formula (X). Will be done. The monomer represented by the following formula (X) can be used, for example, as an amidite for automatic nucleic acid synthesis, and can be applied to, for example, a general nucleic acid automatic synthesis device. The present invention also provides a monomer (amidite) for RNA synthesis represented by the following formula (X).
[0123]
[Chemical 21]

[0124]
　In the formula (X), the description of the formula (IV) can be incorporated for the same part as the above formula (IV). Specifically, in the formula (X), R 2 is an alkylene chain having n carbon atoms. Here, the alkylene chain R 2 optional hydrogen atoms on the carbon atoms of the may be substituted with any substituent or may be unsubstituted. n is not particularly limited and can be appropriately set according to the desired length of the linker Lx 2. However, for example, n is preferably an integer of 1 to 30 from the viewpoint of manufacturing cost, yield, and the like, and more preferably. It is an integer of 1 to 20, and more preferably an integer of 1 to 15. In formula (X), p is 1 or 2, preferably 1. In formula (X), R 3 is a carboxylic acid protecting group. For carboxylic acid protecting groups, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. Specific examples of the carboxylic acid protecting group include DMTr group, Tr group, TBDMS group, ACE group, TOM group, CEE group, CEM group, CE group, TEM group and 2,4-dimethoxybenzyl group. A CE group or a 2,4-dimethoxybenzyl group is preferred.
[0125]
　R 4 is an alkyl group substituted with an electron-withdrawing group, and specific examples thereof include a 2-cyanoethyl group (CE group) and a nitrophenyl ethyl group, and a 2-cyanoethyl group (CE group). Is preferable.
[0126]
　R 5 and R 6 are independently alkyl groups and may be the same or different. Specific examples of -NR 5 R 6 include a diisopropylamino group, a diethylamino group, an ethylmethylamino group and the like, but a diisopropylamino group is preferable.
[0127]
　In a preferred embodiment, the monomer represented by the formula (X) may be represented by the following formula (XI) or the following formula (XVIII).
[0128]
[Chemical 22]

[0129]
[Chemical 23]

[0130]
　The monomer represented by the formula (XI) may be an optically active substance represented by the following formula (XI-1) or the following formula (XI-2).
[0131]
[Chemical 24]

[0132]
[Chemical 25]

[0133]
The monomer represented by the formula (XVIII) may be
an optically active substance represented by the following formula (XVIII-1) or the following formula (XVIII-2).
[0134]
[Chemical 26]

[0135]
[Chemical 27]

[0136]
　An example of a method for synthesizing the monomer represented by the formula (X) will be described below, but as the starting material and the reagent used for the synthesis, a commercially available product may be used as it is, or the monomer is synthesized by a known method. May be good.
[0137]
　The monomer represented by the formula (X) is, for example, a phosphorylation reaction of an alcohol derivative represented by the formula (XII) with a phosphorylating reagent represented by the formula (XIII) as shown in Scheme 1 below. Can be synthesized.
[0138]
[Chemical 28]

[In the formula, X is halogen or NR 5 R 6 , and R 2 , R 3 , R 4 , R 5 , R 6 , and p are the same as the above definitions. ]
[0139]
　Specific examples of the phosphorylation reagent represented by the formula (XIII) include 2-cyanoethyl-N, N-diisopropylchlorophosphoroamidite, nitrophenylethyl-N, N-diisopropylchlorophosphoroamidite, and methyl-N. , N-diisopropylchlorophosphoroamidite or 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphoroamidite and the like.
[0140]
　The equivalent of the phosphorylation reagent represented by the formula (XIII) is preferably 1 to 10 equivalents, more preferably 1.1 to 1.5 equivalents, relative to the alcohol derivative represented by the formula (XII).
[0141]
　Examples of the activator used in the phosphorylation reaction include tetrazole and diisopropylamine tetrazole salt, and diisopropylamine tetrazole salt is preferable.
[0142]
　The equivalent of the activator is preferably 1 to 10 equivalents, more preferably 1.1 to 1.5 equivalents, relative to the alcohol derivative represented by the formula (XII).
[0143]
　Examples of the solvent used for the phosphorylation reaction include tetrahydrofuran, dichloromethane, chloroform, diethyl ether, acetonitrile and the like, but acetonitrile is preferable.
[0144]
　The reaction temperature of the phosphorylation reaction is preferably 20 to 50 ° C, more preferably 20 to 30 ° C. The reaction time of the phosphorylation reaction is preferably 1 to 48 hours, more preferably 2 to 5 hours.
[0145]
　The alcohol derivative represented by the formula (XII) is produced by, for example, as shown in Scheme 2 below, by a condensation reaction between the amine derivative represented by the formula (XIV) and the carboxylic acid derivative represented by the formula (XV). Can be synthesized.
[0146]
[Chemical 29]

[In the formula, R 2 , R 3 , and p are the same as the above definitions. ]
[0147]
　The equivalent of the carboxylic acid derivative represented by the formula (XV) used in the condensation reaction is preferably 1 to 3 equivalents, more preferably 1.1 to 1.5 equivalents, relative to the amine derivative represented by the formula (XIV). preferable.
[0148]
　Examples of the condensing agent used in the condensation reaction include cyclohexylcarbodiimide, N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, benzotriazole-1-yloxy-trisdimethylaminophosphonium salt, and O- (benzo). Triazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate or O- (7-azabenzotriazole-1-yl) -N, N, N', N'- Examples thereof include tetramethyluronium hexafluorophosphate, but N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride is preferable.
[0149]
　The equivalent of the condensing agent is preferably 1 to 10 equivalents, more preferably 1.1 to 1.5 equivalents, relative to the carboxylic acid derivative represented by the formula (XV).
[0150]
　Examples of the base used in the condensation reaction include organic bases such as diisopropylethylamine, triethylamine, pyridine and N-methylmorpholine, and organic acid salts such as potassium carbonate, sodium carbonate or sodium hydrogencarbonate, and diisopropylethylamine or triethylamine may be used. preferable.
[0151]
　The equivalent of the base is preferably 0 to 10 equivalents, more preferably 1 to 3 equivalents, relative to the carboxylic acid derivative represented by the formula (XV).
[0152]
　Examples of the solvent used in the condensation reaction include N, N-dimethylformamide, tetrahydrofuran, dichloromethane, chloroform, diethyl ether and the like, but dichloromethane is preferable.
[0153]
　The reaction temperature of the condensation reaction is preferably 0 to 50 ° C, more preferably 0 to 30 ° C. The reaction time of the condensation reaction is preferably 1 to 48 hours, more preferably 2 to 5 hours.
[0154]
　The amine derivative represented by the formula (XIV) is, for example, as shown in the following scheme 3, the reaction of introducing the protecting group into the carboxylic acid of the amino acid derivative represented by the formula (XVI) and the obtained formula (XVII). ) De-Boc conversion reaction of the amino acid derivative or as shown in Scheme 4, the reaction of introducing the protecting group into the carboxylic acid of the amino acid derivative represented by the formula (XIX) and the obtained formula (XX). It can be synthesized by a de-Fmocization reaction of the amino acid derivative to be produced.
[0155]
Formula 30]

wherein, R 3 , p is same as defined above. ]
[0156]
Formula 31]

wherein, R 3 , p is same as defined above. ]
[0157]
　The protecting group-introducing reaction into the carboxylic acid shall be carried out by a known method described in, for example, the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.). Can be done.
[0158]
　The de-Boc reaction and the de-Fmoc reaction can be carried out, for example, by a known method described in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.). it can.
[0159]
　The amino acid derivative represented by the formula (XVI) or the formula (XIX) can be purchased, or can be produced by a known method or a method similar thereto.
[0160]
　In another embodiment, a monomer for RNA synthesis represented by the following formula (X') can also be used for linking the linker Lx 2 to the 5'end of X. This monomer basically corresponds to the linker represented by the above formula (IV'), and the description of the linker represented by the above formula (IV') is described by the monomer represented by the following formula (X'). It is also used for. The present invention also provides a monomer (amidite) for RNA synthesis represented by the following formula (X').
[0161]
[Chemical 32]

[0162]
　In the formula (X'), the description of the formula (IV') can be incorporated for the same part as the above formula (IV'). Specifically, in the formula (X'), R 2 is an alkylene chain having n carbon atoms. Here, the alkylene chain R 2 optional hydrogen atoms on the carbon atoms of the may be substituted with any substituent or may be unsubstituted. n is not particularly limited and can be appropriately set according to the desired length of the linker Lx 2. However, for example, n is preferably an integer of 1 to 30 from the viewpoint of manufacturing cost, yield, and the like, and more preferably. It is an integer of 1 to 20, and more preferably an integer of 1 to 15. In formula (X'), R 3 is a carboxylic acid protecting group. For carboxylic acid protecting groups, for example, the description in the literature (Protective Groups in Organic Synthesis 4th Edition, by Greene et al., 2007, John Wiley & Sons, Inc.) can be incorporated. Specific examples of the carboxylic acid protecting group include DMTr group, Tr group, TBDMS group, ACE group, TOM group, CEE group, CEM group, CE group, TEM group and 2,4-dimethoxybenzyl group. CE groups are preferred.
[0163]
　R 4 is an alkyl group substituted with an electron-withdrawing group, and specific examples thereof include a 2-cyanoethyl group (CE group) and a nitrophenyl ethyl group, and a 2-cyanoethyl group (CE group). Is preferable.
[0164]
　R 5 and R 6 are independently alkyl groups and may be the same or different. Specific examples of -NR 5 R 6 include a diisopropylamino group, a diethylamino group, an ethylmethylamino group and the like, but a diisopropylamino group is preferable.
[0165]
　In a preferred embodiment, the monomer represented by the formula (X') may be represented by the following formula (XXIII).
[0166]
[Chemical 33]

[0167]
　The monomer represented by the formula (X') is the same as in Schemes 1 to 3 above, for example, using an amino acid derivative represented by the formula (XVI') instead of the amino derivative represented by the formula (XVI). It can be manufactured by the method of.
[0168]
[Chemical 34]

[0169]
　The amino acid derivative represented by the formula (XVI') can be purchased, or can be produced by a known method or a method similar thereto.
[0170]
　Further, for linking the linker Ly to the 5'end of Yc, any suitable RNA synthesis monomer, for example, is described in the literature (Japanese Patent No. 5261677 or Japanese Patent No. 5876890). Known known RNA synthesis monomers can be used.
[0171]
　In the hairpin-type positive-strand RNA molecule produced by the method of the present invention, XY comprises a gene expression-suppressing sequence for a target gene. The gene expression-suppressing sequence may be contained only in X or only Y. The gene expression-suppressing sequence is preferably a sense sequence or antisense sequence of all or part of the mRNA transcribed from the target gene.
[0172]
　The hairpin-type positive-strand RNA molecule may contain one gene expression-suppressing sequence or two or more. The hairpin-type single-stranded RNA molecule may have, for example, two or more of the same expression-suppressing sequences for the same target gene, two or more different expression-suppressing sequences for the same target, or different It may have two or more different expression-suppressing sequences for the target gene. A hairpin-type positive-strand RNA molecule having two or more gene expression-suppressing sequences for different target genes is useful for suppressing the expression of two or more different target genes. In the present invention, the “gene” refers to a genomic region transcribed into mRNA, which may be a protein coding region or an RNA coding region.
[0173]
　The hairpin-type single-strand RNA molecule according to the present invention has an ability to suppress the expression of a target gene via a gene expression-suppressing sequence. The suppression of the expression of the target gene by the hairpin-type positive-strand RNA molecule according to the present invention is preferably due to RNA interference, but is not limited thereto. In RNA interference, a long double-stranded RNA (dsRNA) is generally cleaved in a cell by a Dicer into a short double-stranded RNA (siRNA: small interfering RNA) having a protruding 3'end of about 19 to 21 base pairs. This is a phenomenon in which one of the single-stranded RNAs binds to the target mRNA and decomposes the target mRNA to suppress the translation of the target mRNA, thereby suppressing the expression of the target gene from which the target mRNA is derived. .. Various types of single-stranded RNA sequences contained in siRNA that bind to target mRNA have been reported, for example, depending on the type of target gene. In the present invention, for example, a sequence of single-strand RNA contained in siRNA can be used as a gene expression-suppressing sequence. The hairpin-type single-strand RNA molecule produced by the method of the present invention can suppress the expression of a target gene by being cleaved in vivo to generate siRNA. The hairpin-type positive-strand RNA molecule according to the present invention can be used for the treatment or prevention of diseases or disorders associated with the expression or increased expression of a target gene.
[0174]
　The gene expression-suppressing sequence is preferably 19 to 30 bases in length, more preferably 19 to 27 bases in length, and may be, for example, 19, 20, 21, 22, or 23 bases in length. The gene expression-suppressing sequence preferably consists of an RNA sequence that is completely identical or completely complementary to the sequence of at least a part of the mRNA of the target gene.
[0175]
　The target gene is not particularly limited, and examples thereof include a TGF-β1 gene, a GAPDH gene, a LMNA1 gene, and an LMNA gene. When the target gene is the TGF-β1 gene, the hairpin-type single-strand RNA molecule produced by the method of the present invention suppresses the expression of the TGF-β1 gene in vivo. Such hairpin-type single-stranded RNA molecules treat or prevent diseases or disorders associated with TGF-β1 gene expression or increased expression, such as pulmonary fibrosis and acute lung disease, through suppression of TGF-β1 gene expression. Can be used for.
[0176]
　One example of a hairpin-type single-stranded RNA molecule containing a gene expression-suppressing sequence produced by the method of the present invention has the base sequence represented by SEQ ID NO: 1 and the base sequence represented by SEQ ID NO: 2. It is obtained by a combination of the first single-stranded oligo RNA molecule (strand A) having the above and the second single-stranded oligo RNA molecule (strand B) having the base sequence represented by SEQ ID NO: 3. The nucleotide sequence represented by SEQ ID NO: 1 includes a gene expression-suppressing sequence for the TGF-β1 gene, which is a target gene. The sequences at positions 29 to 47 of the nucleotide sequence represented by SEQ ID NO: 1 correspond to the gene expression-suppressing sequence (active sequence) (FIG. 2).
[0177]
　Further, as another example, the first single-stranded oligo RNA molecule (strand C) having the base sequence represented by SEQ ID NO: 4 and having the base sequence represented by SEQ ID NO: 5 and SEQ ID NO: 6 It is obtained by a combination of a second single-stranded oligo RNA molecule (strand D) having the indicated base sequence. The nucleotide sequence represented by SEQ ID NO: 4 contains a gene expression-suppressing sequence for the GAPDH gene, which is a target gene. The sequences 27 to 45 of the nucleotide sequence represented by SEQ ID NO: 4 correspond to the gene expression suppression sequence (active sequence).
[0178]
　In the present invention, the first single-strand oligo RNA molecule represented by the above formula (I) and the second single-strand oligo RNA molecule represented by the above formula (II) are mixed in a reaction solvent. the presence of a dehydrating condensing agent (also condensing agent referred.), is reacted, the linker Lx 1 and linker Lx 2 by forming an amide bond between, to manufacture a single-stranded RNA molecule the hairpin it can. The reaction solvent is a mixed solvent containing a buffer solution and a hydrophilic organic solvent.
[0179]
　Examples of the buffer solution include 2- (N-morpholino) ethanesulfonic acid buffer solution (hereinafter, MES buffer solution), 3- (N-morpholino) -2-hydroxypropanesulfonic acid buffer solution (hereinafter, MOPSO buffer solution), and the like. Piperazin-1,4-bis (2-ethanesulfonic acid) buffer (hereinafter, PIPES buffer), 3- (N-morpholino) propanesulfonic buffer (hereinafter, MOPS buffer), N, N-bis (hereinafter, MOPS buffer) 2-Hydroxyethyl) -2-aminoethanesulfonic buffer (BES buffer), 3- [N, N-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic buffer (hereinafter, BES buffer) DIPSO buffer), 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid buffer (hereinafter, HEPES buffer), piperazin-1,4-bis (2-hydroxypropanesulfonic acid) buffer Liquid (hereinafter, POPSO buffer), 4- (2-hydroxyethyl) piperazin-1- (2-hydroxypropane-3-sulfonic acid buffer (hereinafter, HEPPSO buffer)), 4- (2-hydroxyethyl)- Examples thereof include, but are not limited to, 1-piperazin propanesulfonic acid buffer (hereinafter referred to as HEPPS buffer), and MES buffer or MOPS buffer is preferably used.
[0180]
　The pH of the buffer can be adjusted as appropriate, but is preferably 6.5 to 7.5, more preferably 6.9 to 7.5, for example 6.5 to 7.0, 6. It is 9.9 to 7.1, or 7.0 to 7.5.
[0181]
　In the present invention, a hydrophilic organic solvent can be used as a component of the reaction solvent. The hydrophilic organic solvent may be, for example, a hydrophilic aprotic organic solvent.
[0182]
　The hydrophilic organic solvent means an organic solvent that can be mixed with water in any ratio, and is a sulfone-based solvent such as sulfolane, a sulfoxide-based solvent such as dimethyl sulfoxide, and an amide such as N, N-dimethylformamide and N-methylpyrrolidone. System solvents, urea solvents such as N, N'-dimethylethylene urea, nitrile solvents such as acetonitrile, ether solvents such as tetrahydrofuran and 1,2-dimethoxyethane, alcohol solvents such as ethanol and tert-butyl alcohol Can be mentioned. The "aprotonic" organic solvent means an organic solvent having no proton donating property, and the hydrophilic aprotonic organic solvent includes a sulfone-based solvent such as sulfolane, a sulfoxide-based solvent such as dimethyl sulfoxide, and the like. Amid solvents such as N, N-dimethylformamide and N-methylpyrrolidone, urea solvents such as N, N'-dimethylethyleneurea, nitrile solvents such as acetonitrile, ether solvents such as tetrahydrofuran and 1,2-dimethoxyethane. Examples of the solvent include dimethyl sulfoxide (hereinafter, DMSO), N, N-dimethylformamide (hereinafter, DMF), N, N'-dimethylethyleneurea (hereinafter, DMEU), or acetonitrile, and DMSO or DMF is more preferable. preferable.
[0183]
　A dehydration condensing agent, an N-hydroxy nitrogen-containing aromatic compound (for example, hydroxybenzotriazole or a derivative thereof), a cyano (hydroxyimino) acetate, or an N-hydrocarbon-substituted imidazole derivative is added by dissolving or suspending it in an organic solvent. In the case, the organic solvent shall be included in the "organic solvent" in the mixed solvent. One kind of organic solvent may be used, or two or more kinds may be used in combination.
[0184]
　In one embodiment, the ratio of the organic solvent in the mixed solvent which is the reaction solvent is 15 to 70% by volume (v / v%) of the total amount of the solvent (the total amount of the mixed solvent including the buffer solution and the organic solvent). It may be, for example, 20 to 70% by volume, 20 to 65% by volume, 35 to 60% by volume, 35 to 55% by volume, or 50 to 55% by volume. In the present invention, a dehydration condensing agent, an N-hydroxy nitrogen-containing aromatic compound (for example, hydroxybenzotriazole or a derivative thereof), a cyano (hydroxyimino) acetate or an N-hydrocarbon-substituted imidazole derivative is used in water, a buffer or an organic solvent. When added by dissolving or suspending in a solution such as, the total amount of the solvent is a dehydration condensing agent, an N-hydroxy nitrogen-containing aromatic compound (for example, hydroxybenzotriazole or a derivative thereof), a cyano (hydroxyimino) acetate or N-. Includes the amount of solution containing the hydrocarbon-substituted imidazole derivative. Also, in calculating the ratio of the organic solvent in the mixed solvent, a dehydration condensing agent, an N-hydroxy nitrogen-containing aromatic compound (for example, hydroxybenzotriazole or a derivative thereof), a cyano (hydroxyimino) acetate, or an N-hydrocarbon-substituted imidazole. When the derivative is dissolved or suspended in an organic solvent and added, the amount of the organic solvent shall be included in the total amount of the organic solvent in the mixed solvent.
[0185]
　When the hydrophilic organic solvent is DMSO, the ratio of DMSO in the mixed solvent is preferably 20 to 65% by volume, preferably 35 to 60% by volume, and 50 to 55 or 50 to 50% by volume of the total amount of the solvent. More preferably, it is 60% by volume. When the hydrophilic organic solvent is DMSO and the dehydration condensing agent is a uronium type dehydration condensing agent containing an N-hydroxy nitrogen-containing aromatic ring structure, for example, a benzotriazolyl uronium type dehydration condensing agent such as HATU, the mixture is mixed. The ratio of DMSO in the solvent is preferably 35 to 65% by volume, more preferably 50 to 65% by volume, and may be, for example, 50 to 60% by volume of the total amount of the solvent. When the hydrophilic organic solvent is DMSO and the dehydration condensing agent is a triazine type dehydration condensing agent, for example, a triazine type quaternary morpholinium derivative such as DMT-MM, the ratio of DMSO in the mixed solvent is 35 of the total amount of the solvent. It is preferably from to 65% by volume, more preferably from 35 to 60% by volume, and may be, for example, 50 to 60% by volume or 50 to 55% by volume. In these cases, the pH of the reaction solvent (mixed solvent) is not limited to the following, but is preferably 6.5 to 7.5, more preferably 6.9 to 7.5. For example, it may be 6.5 to 7.0, 6.9 to 7.1, or 7.0 to 7.5.
[0186]
　When the hydrophilic organic solvent is DMF, the ratio of DMF in the mixed solvent is preferably 20 to 65% by volume, preferably 35 to 65% by volume, and 50 to 65% by volume of the total amount of the solvent. More preferably. When the hydrophilic organic solvent is DMF and the dehydration condensing agent is a uronium type dehydration condensing agent containing an N-hydroxy nitrogen-containing aromatic ring structure, for example, a benzotriazolyl uronium type dehydration condensing agent such as HATU, the mixture is mixed. The ratio of DMF in the solvent is preferably 35 to 65% by volume, more preferably 50 to 65% by volume, and may be, for example, 50 to 60% by volume of the total amount of the solvent. In these cases, the pH of the reaction solvent (mixed solvent) is not limited to the following, but is preferably 6.5 to 7.5, more preferably 6.9 to 7.5. For example, it may be 6.5 to 7.0, 6.9 to 7.1, or 7.0 to 7.5.
[0187]
　When the hydrophilic organic solvent is DMEU, the ratio of DMEU in the mixed solvent is preferably 20 to 65% by volume, preferably 35 to 65% by volume, and 50 to 60% by volume of the total amount of the solvent. More preferably. When the hydrophilic organic solvent is DMEU and the dehydration condensing agent is a uronium type dehydration condensing agent containing an N-hydroxy nitrogen-containing aromatic ring structure, for example, a benzotriazolyl uronium type dehydration condensing agent such as HATU, the mixture is mixed. The ratio of DMEU in the solvent is preferably 35 to 65% by volume, more preferably 50 to 65% by volume, and may be, for example, 50 to 60% by volume of the total amount of the solvent. In these cases, the pH of the reaction solvent (mixed solvent) is not limited to the following, but is preferably 6.5 to 7.5, more preferably 6.9 to 7.5. For example, it may be 6.5 to 7.0, 6.9 to 7.1, or 7.0 to 7.5.
[0188]
　When the hydrophilic organic solvent is acetonitrile, the ratio of acetonitrile in the mixed solvent is preferably 20 to 65% by volume, preferably 35 to 65% by volume, and 50 to 65% by volume of the total amount of the solvent. More preferably. When the hydrophilic organic solvent is acetonitrile and the dehydration condensing agent is a uronium type dehydration condensing agent containing an N-hydroxy nitrogen-containing aromatic ring structure, for example, a benzotriazolyl uronium type dehydration condensing agent such as HATU, the mixture is mixed. The ratio of acetonitrile in the solvent is preferably 35 to 65% by volume, more preferably 50 to 65% by volume, and may be, for example, 50 to 60% by volume of the total amount of the solvent. In these cases, the pH of the reaction solvent (mixed solvent) is not limited to the following, but is preferably 6.5 to 7.5, more preferably 6.9 to 7.5. For example, it may be 6.5 to 7.0, 6.9 to 7.1, or 7.0 to 7.5.

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 is represented by the following formula (I) in a mixed solvent containing a buffer solution and a hydrophilic organic solvent in the presence of a dehydration condensing agent. The step of reacting the first single-stranded oligo RNA molecule to be subjected to the second single-stranded oligo RNA molecule represented by the following formula (II) is included, and
　5'-Xc-Lx 1　... (I). )
　Lx 2 -X-Y-Ly-Yc-3 '· · · (II)
[in the formula (I) and formula (II), X, Xc, Y and Yc consists ribonucleotide residues, Xc is , X is complementary, Yc is complementary to Y, Ly is a non-nucleotide linker, Lx 1 is a non-nucleotide linker with an amino group, and Lx 2 is a carboxyl group. It is a non-nucleotide linker having, and XY contains a gene expression-suppressing sequence for the target gene.] The
　dehydration condensing agent is a triazine-type dehydration condensing agent and an uronium-type dehydration condensation containing an N-hydroxy nitrogen-containing aromatic ring structure. It is selected from the group consisting of an agent, a carbodiimide type dehydration condensing agent, a 2-halopyridinium type dehydration condensing agent, and a formamidinium type dehydration condensing agent, and
　when the dehydration condensing agent is a carbodiimide type dehydration condensing agent, it contains N-hydroxy. Used in combination with nitrogen aromatic compounds or cyano (hydroxyimino) acetate,
　When the dehydration condensing agent is a 2-halopyridinium type dehydration condensing agent, it is used in combination with an N-hydroxy nitrogen-containing aromatic compound, and
　when the dehydration condensing agent is a formamidinium type dehydration condensing agent, N-hydroxy A
method for producing a hairpin-type single-stranded RNA molecule , which is used in combination with a nitrogen-containing aromatic compound or an N-hydrocarbon-substituted imidazole derivative .
[Claim 2]
The production method according to claim 1 　, wherein the linker Ly is a non-nucleotide linker having an amino acid skeleton or an amino alcohol skeleton, and the linker Lx 2 is a non-nucleotide linker having an amino acid skeleton.
[Claim 3]
　The Ly is a non-nucleotide linker containing at least one of a pyrrolidine skeleton and a piperidine skeleton, or a non-nucleotide linker containing -NHCH 2 COO-, and the Lx 2 is at least a pyrrolidine skeleton having a carboxyl group and a piperidine skeleton. The production method according to claim 1 or 2, which is a non-nucleotide linker containing one or a non-nucleotide linker containing -NHCH 2 COOH.
[Claim 4]
The production method according to any one of claims 1 to 3, wherein 　Lx 1 is represented by the following formula (III) and Lx 2 is represented by the following formula (IV) or the following formula (IV').
[Chemical

formula 1] [In formula (III), R 1 is an optionally substituted alkylene chain, and -OR 1 is attached to the 3'end of Xc via a phosphodiester bond]
[Chemical formula 1] 2]

[In formula (IV), R 2 is an optionally substituted alkylene chain, p is 1 or 2, and -OR 2 is a phosphodiester bond at the 5'end of X. In formula (IV'), R 2 is an optionally substituted alkylene chain and -OR 2 is attached to the 5'end of X via a phosphodiester bond].
[Claim 5]
　(I) The uronium-type dehydration-condensing agent containing the N-hydroxy nitrogen-containing aromatic ring structure is a benzotriazolyluronium-type dehydration-condensation agent.
　(Ii) The N-hydroxy nitrogen-containing aromatic compound is hydroxybenzo. Triazole or a derivative thereof,
　(iii) said cyano (hydroxyimino) acetate is a cyano (hydroxyimino) alkyl ester, and / or
　(iv) said N-hydrocarbon substituted imidazole derivative is N-alkyl.
The production method according to any one of claims 1 to 4 , which is an imidazole derivative .
[Claim 6]
　The production method according to any one of claims 1 to 5, wherein the hydrophilic organic solvent is a hydrophilic aprotic organic solvent.
[Claim 7]
　The production method according to claim 6, wherein the hydrophilic aprotic organic solvent is dimethyl sulfoxide, N, N-dimethylformamide, N, N'-dimethylethyleneurea, or acetonitrile.
[Claim 8]
　The dehydration condensing agent is 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride, O- (7-azabenzotriazole-1-yl)-. N, N, N', N'-tetramethyluronium hexafluorophosphate, N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, 2-chloro-1-methylpyridinium iodide, or Chloro-N, N, N', N'-tetramethylform amidineium hexafluorophosphate, the
N-hydroxy nitrogen-containing aromatic compound is 1-hydroxy-7-azabenzotriazole, and the
cyano (
The production method according to any one of claims 1 to 7 , wherein the hydroxyimino) acetate ester is ethyl cyano (hydroxyimino) acetate, and the N-hydrocarbon-substituted imidazole derivative is N-methylimidazole.
[Claim 9]
　The combination of the dehydration condensing agent and the hydrophilic aprotic organic solvent is O- (7-azabenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate. And dimethyl sulfoxide, combination of O- (7-azabenzotriazole-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate and N, N-dimethylformamide, Combination of N- (3'-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride and 1-hydroxy-7-azabenzotriazole and dimethyl sulfoxide, or chloro-N, N, N', N'-tetramethyl The production method according to any one of claims 7 or 8, which is a combination of formamidinium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole and dimethyl sulfoxide.
[Claim 10]
　The production method according to any one of claims 1 to 9, wherein the pH of the buffer solution is 6.5 to 7.5.
[Claim 11]
　Ly is a non-nucleotide linker containing a pyrrolidine skeleton or a piperidine skeleton, Lx 1 is a non-nucleotide linker having an amino group, and Lx 2 is at least one of a pyrrolidine skeleton and a piperidine skeleton having a carboxyl group. The production method according to any one of claims 1 to 10, which is a non-nucleotide linker comprising the above.
[Claim 12]
　The production method according to any one of claims 1 to 11, wherein Ly is represented by the following formula (V).
[Chemical 3]

[Claim 13]
The production method according to any one of claims 1 to 12, wherein 　Lx 1 is represented by the following formula (VI) and Lx 2 is represented by the following formula (VII).
[Chemical 4]

[Chemical 5]

[Claim 14]
　The production method according to any one of claims 1 to 13, wherein the target gene is a TGF-β1 gene.
[Claim 15]
　The production method according to any one of claims 1 to 14, wherein the hairpin-type single-strand RNA molecule comprises the base sequence represented by SEQ ID NO: 1.
[Claim 16]
　The single-strand oligo RNA molecule according to (a) or (b) below.
(A) 1 th ribonucleotide residues Lx 2 is connected to the 26 th and 27 th single strand consisting of the nucleotide sequence of ribonucleotide residues represented by SEQ ID NO: 3 which are connected via a Ly oligo RNA molecule
(b) 1 th ribonucleotide residues Lx 2 is connected with, made of 26 th and 27 th nucleotide sequence ribonucleotide residues represented by SEQ ID NO: 6 which is connected via a Ly Single-stranded oligo RNA molecule
[Claim 17]
　A kit for producing a hairpin-type single-stranded RNA molecule for suppressing the expression of a target gene, which comprises the combination of single-stranded oligo RNA molecules according to (1) or (2) below.
(1) The first single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 2 in which the 24th ribonucleotide residue is linked to Lx 1 , and the first ribonucleotide residue is Lx 2. Combination of the second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 3, in which the 26th and 27th ribonucleotide residues are linked via Ly
(2) 22nd ribonucleotide residues Lx of 1 and the first single-stranded oligo RNA molecule comprising the nucleotide sequence represented by SEQ ID NO: 5 which is connected with, first ribonucleotide residues Lx 2 is connected with, 26 A combination of a second single-stranded oligo RNA molecule consisting of the nucleotide sequence represented by SEQ ID NO: 6, in which the 27th and 27th ribonucleotide residues are linked via Ly.