Method of producing hairpin single-stranded RNA molecules

A method of producing a hairpin single-stranded RNA molecule capable of inhibiting expression of a target gene, comprising:

• reacting a first single-stranded oligo-RNA molecule represented by formula (I) with a second single-stranded oligo-RNA molecule represented by formula (II) in a mixed solvent comprising a buffer solution and a hydrophilic organic solvent in the presence of a dehydration condensation agent:
• 5′-Xc-Lx1 (I)
• Lx2-X—Y-Ly-Yc-3′ (II)
• wherein, in formula (I) or (II), X, Xc, Y and Yc each is composed of a ribonucleotide residue, Xc is complementary to X, Yc is complementary to Y, Ly is a non-nucleotide linker, Lx1 is a non-nucleotide linker having an amino group, Lx2 is a non-nucleotide linker having a carboxyl group, and X—Y comprises a gene expression-inhibiting sequence for the target gene,
• the dehydration condensation agent is selected from the group consisting of a triazine-based dehydration condensation agent, a uronium-based dehydration condensation agent comprising an N-hydroxy nitrogen-containing aromatic ring structure, a carbodiimide-based dehydration condensation agent, a 2-halopyridinium-based dehydration condensation agent, and a formamidinium-based dehydration condensation agent,
• if the dehydration condensation agent is the carbodiimide-based dehydration condensation agent, the dehydration condensation agent is used in combination with an N-hydroxy nitrogen-containing aromatic compound or a cyano(hydroxyimino)acetic acid ester,
• if the dehydration condensation agent is the 2-halopyridinium-based dehydration condensation agent, the dehydration condensation agent is used in combination with an N-hydroxy nitrogen-containing aromatic compound, and
• if the dehydration condensation agent is the formamidinium-based dehydration condensation agent, the dehydration condensation agent is used in combination with an N-hydroxy nitrogen-containing aromatic compound or an N-hydrocarbon-substituted imidazole derivative.