Method of producing hairpin single-stranded RNA molecules

Patent metadata

Field	Value
Publication number	US-11891602-B2
Application number	US-201917276194-A
Country	US
Kind code	B2
Filing date	Oct 2, 2019
Priority date	Oct 2, 2018
Publication date	Feb 6, 2024
Grant date	Feb 6, 2024

Abstract

A method produces a hairpin single-stranded RNA molecule capable of inhibiting expression of a target gene, including the step of 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 including a buffer solution and a hydrophilic organic solvent in the presence of a dehydration condensation agent: 5′-Xc-Lx1 (I) and Lx2-X—Y-Ly-Yc-3′ (II), wherein the dehydration condensation agent is selected from the group consisting of a triazine-based dehydration condensation agent, a uronium-based dehydration condensation agent including 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.

First claim

The invention claimed is:

1. 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-Lx 1   (I)

Lx 2 -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, Lx 1 is a non-nucleotide linker having an amino group, Lx 2 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.

2. The method according to claim 1 , wherein the linker Ly is a non-nucleotide linker having an amino acid backbone or an amino alcohol backbone, and the linker Lx 2 is a non-nucleotide linker having an amino acid backbone.

3. The method according to claim 1 , wherein the Ly is a non-nucleotide linker comprising at least one of a pyrrolidine backbone or a piperidine backbone, or a non-nucleotide linker comprising —NHCH 2 COO—, and the Lx 2 is a non-nucleotide linker comprising at least one of a pyrrolidine backbone or a piperidine backbone and having a carboxyl group, or a non-nucleotide linker comprising —NHCH 2 COOH.

4. The method according to claim 1 , wherein Lx 1 is represented by formula (III):

wherein, in formula (III), R 1 is an optionally substituted alkylene chain, and —OR′ is bonded to the 3′ end of Xc via a phosphodiester bond, and
Lx 2 is represented by formula (IV) or formula (IV′):

wherein, in formula (IV), R 2 is an optionally substituted alkylene chain, p is 1 or 2, and —OR 2 is bonded to the 5′ end of X via a phosphodiester bond; and in formula (IV′), R 2 is an optionally substituted alkylene chain, and —OR 2 is bonded to the 5′ end of X via a phosphodiester bond.

5. The method according to claim 1 , wherein at least one of:
(i) the uronium-based dehydration condensation agent comprising an N-hydroxy nitrogen-containing aromatic ring structure is a benzotriazolyluronium-based dehydration condensation agent,
(ii) the N-hydroxy nitrogen-containing aromatic compound is hydroxybenzotriazole or a derivative thereof,
(iii) the cyano(hydroxyimino)acetic acid ester is a cyano(hydroxyimino)acetic acid alkyl ester, and
(iv) the N-hydrocarbon-substituted imidazole derivative is an N-alkylimidazole derivative.

6. The method according to claim 1 , wherein the hydrophilic organic solvent is a hydrophilic aprotic organic solvent.

7. The method according to claim 6 , wherein the hydrophilic aprotic organic solvent is dimethyl sulfoxide, N,N-dimethylformamide, N,N′-dimethylethyleneurea, or acetonitrile.

8. The method according to claim 1 ,
wherein the dehydration condensation agent is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-fluoroophosphate, N-(3′-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 2-chloro-1-methylpyridinium iodide, or chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate,
the N-hydroxy nitrogen-containing aromatic compound is 1-hydroxy-7-azabenzotriazole,
the cyano(hydroxyimino)acetic acid ester is ethyl cyano(hydroxyimino)acetate, and
the N-hydrocarbon-substituted imidazole derivative is N-methylimidazole.

9. The method according to claim 7 , wherein a combination of the dehydration condensation agent and the hydrophilic aprotic organic solvent is a combination of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and dimethyl sulfoxide, a combination of O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate and N,N-dimethylformamide, a combination of N-(3′-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 1-hydroxy-7-azabenzotriazole and dimethyl sulfoxide, or a combination of chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate, 1-hydroxy-7-azaben-zotriazole and dimethyl sulfoxide.

10. The method according to claim 1 , wherein pH of the buffer solution is 6.5 to 7.5.

11. The method according to claim 1 , wherein Ly is a non-nucleotide linker comprising a pyrrolidine backbone or a piperidine backbone, Lx 1 is a non-nucleotide linker having an amino group, and Lx 2 is a non-nucleotide linker comprising at least one of a pyrrolidine backbone or a piperidine backbone and having a carboxyl group.

12. The method according to claim 1 , wherein Ly is represented by formula (V):

13. The method according to claim 1 , wherein Lx 1 is represented by formula (VI):

and Lx 2 is represented by formula (VII):

14. The method according to claim 1 , wherein the target gene is TGF-β1 gene.

15. The method according to claim 1 , wherein the hairpin single-stranded RNA molecule comprises the nucleotide sequence set forth in SEQ ID NO: 1.

Assignees

• Toray Industries

Inventors

• Koshimoto Kyohei
• Iseki Katsuhiko
• Inada Hideaki
• Fujita Tatsuya
• Okimura Keiichi
• Kunishima Munetaka
• Ohgi Tadaaki
• Aoki Eriko

Classifications

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2330/30

  chemically synthesised · CPC title

• C12N15/111 Primary

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12N15/1136 Primary

  against growth factors, growth regulators, cytokines, lymphokines or hormones · CPC title