Method of producing hairpin single-stranded RNA molecule

Patent metadata

Field	Value
Publication number	US-11920131-B2
Application number	US-201916981329-A
Country	US
Kind code	B2
Filing date	Mar 29, 2019
Priority date	Mar 30, 2018
Publication date	Mar 5, 2024
Grant date	Mar 5, 2024

Abstract

A method produces a hairpin single-stranded RNA molecule capable of inhibiting expression of a target gene, the method including: (i) an annealing step of annealing a first single-stranded oligoRNA molecule and a second single-stranded oligoRNA molecule; and (ii) a ligation step of ligating 3′ end of the first single-stranded oligoRNA molecule and 5′ end of the second single-stranded oligoRNA molecule by an Rnl2 family ligase, wherein a sequence produced by ligating the first single-stranded oligoRNA molecule and the second single-stranded oligoRNA molecule includes a gene expression-inhibiting sequence for the target gene.

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, the method comprising:
an annealing step of annealing a first single-stranded oligoRNA molecule and a second single-stranded oligoRNA molecule; and
a ligation step of ligating 3′ end of the first single-stranded oligoRNA molecule and 5′ end of the second single-stranded oligoRNA molecule by an Rnl2 family ligase,
wherein the first single-stranded oligoRNA molecule comprises a first RNA region and a second RNA region connected via a first linker, and one of the first RNA region and the second RNA region is capable of complementarily binding to the other,
the second single-stranded oligoRNA molecule comprises a third RNA region and a fourth RNA region connected via a second linker, and one of the third RNA region and the fourth RNA region is capable of complementarily binding to the other,
the first linker and the second linker are non-nucleotide linkers,
the first single-stranded oligoRNA molecule and the second single-stranded oligoRNA molecule are capable of forming an intermolecular double strand between complementary sequences at 5′-end or 3′-end thereof,
when the double strand is formed between the first single-stranded oligoRNA molecule and the second single-stranded oligoRNA molecule during the annealing step, a nick occurs between the 3′-end ribonucleotide residue of the first single-stranded oligoRNA molecule and the 5′-end ribonucleotide residue of the second single-stranded oligoRNA molecule, and a gap of at least one ribonucleotide residue is present between the 5′-end ribonucleotide residue of the first single-stranded oligoRNA molecule and the 3′-end ribonucleotide residue of the second single-stranded oligoRNA molecule, and
a sequence produced by ligating the first single-stranded oligoRNA molecule and the second single-stranded oligoRNA molecule comprises a gene expression-inhibiting sequence for the target gene.

2. The method according to claim 1 , wherein the first single-stranded oligoRNA molecule is represented by formula (I) and the second single-stranded oligoRNA molecule is represented by formula (II):
5′-Xs-Lx 1 -Xa-3′  (I)

5′-Ya 1 -Ya 2 -Ya 3 -Lx 2 -Ys-3′  (II)

wherein Xs, Xa, Ya 1 , Ya 2 , Ya 3 , and Ys each represent one or more ribonucleotide residues,
Lx 1 and Lx 2 represent the first linker and the second linker, respectively,
Ya 3 is complementary to Ys,
Xa-Ya 1 , which is generated by the ligation step, is complementary to Xs, and
Xa-Ya 1 -Ya 2 -Ya 3 , which is generated by the ligation step, comprises a gene expression-inhibiting sequence for the target gene.

3. The method according to claim 1 , wherein the first single-stranded oligoRNA molecule has an uracil (U) or adenine (A) at the 3′ end, and the second single-stranded oligoRNA molecule has an uracil (U) or adenine (A) at the 5′ end.

4. The method according to claim 1 , wherein the first linker and the second linker are each independently a non-nucleotide linker comprising at least one selected from a pyrrolidine backbone and a piperidine backbone.

5. The method according to claim 1 , wherein the Rnl2 family ligase is T4 RNA ligase 2.

6. The method according to claim 1 , wherein the ligating is carried out in a reaction solution at pH 7.4 to 8.6.

7. The method according to claim 1 , wherein the ligating is carried out in a reaction solution comprising 2 to 10 mM divalent metal ion.

8. The method according to claim 1 , wherein the first linker and the second linker are each independently a non-nucleotide linker represented by formula (VI):

9. The method according to claim 1 , wherein the target gene is TGF-β1 gene, GAPDH gene, LAMA1 gene, or LMNA gene.

10. The method according to claim 1 , wherein the hairpin single-stranded RNA molecule consists of the nucleotide sequence set forth in SEQ ID NO: 1 wherein ribonucleotide residues at positions 24 and 25 are connected via the first linker and ribonucleotide residues at positions 50 and 51 are connected via the second linker.

11. The method according to claim 1 , wherein the first single-stranded oligoRNA molecule and the second single-stranded oligoRNA molecule are any of (1) to (21):
(1) a combination of the first single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 7 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 6 in which ribonucleotide residues at positions 10 and 11 are connected via the second linker;
(2) a combination of the first single-stranded oligoRNA molecule consisting of a nucleotide sequence set forth in SEQ ID NO: 19 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 18 in which ribonucleotide residues at positions 16 and 17 are connected via the second linker;
(3) a combination of the first single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 27 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 26 in which ribonucleotide residues at positions 20 and 21 are connected via the second linker;
(4) a combination of the first single-stranded oligoRNA molecule consisting of a nucleotide sequence set forth in SEQ ID NO: 29 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 28 in which ribonucleotide residues at positions 21 and 22 are connected via the second linker;
(5) a combination of the first single-stranded oligoRNA molecule consisting of a nucleotide sequence set forth in SEQ ID NO: 31 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 30 in which ribonucleotide residues at positions 22 and 23 are connected via the second linker;
(6) a combination of the first single-stranded oligoRNA molecule consisting of a nucleotide sequence set forth in SEQ ID NO: 33 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 32 in which ribonucleotide residues at positions 23 and 24 are connected via the second linker;
(7) a combination of the first single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 5 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 4 in which ribonucleotide residues at positions 9 and 10 are connected via the second linker;
(8) a combination of the first single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 9 in which ribonucleotide residues at positions 24 and 25 are connected via the first linker and the second single-stranded oligoRNA molecule consisting of the nucleotide sequence set forth in SEQ ID NO: 8 in which ribonucleotide residues at positions 11 and 12 are conne

Assignees

• Toray Industries

Inventors

• Inada Hideaki
• Iseki Katsuhiko
• Okimura Keiichi
• Sanosaka Masato
• TAKASHINA Ayumi

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

• C12Y605/01003 Primary

  RNA ligase (ATP) (6.5.1.3) · CPC title

• C12N15/00 Primary

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title