Methods and compositions relating to covalently closed nucleic acids

Patent metadata

Field	Value
Publication number	US-12571024-B2
Application number	US-202217820856-A
Country	US
Kind code	B2
Filing date	Aug 18, 2022
Priority date	Aug 19, 2021
Publication date	Mar 10, 2026
Grant date	Mar 10, 2026

Abstract

Provided herein are methods, systems, and compositions for assembly of covalently closed double stranded nucleic acids. Provided herein are methods, systems, and compositions for assembly covalently closed double stranded nucleic acids for use in various downstream processes.

First claim

What we claim is:

1 . A method for assembly of a covalently closed double stranded nucleic acid, comprising:
(a) providing a double stranded nucleic acid; (b) amplifying the double stranded nucleic acid using a primer comprising one or more uracils to generate a double stranded nucleic acid comprising one or more uracils at a 5′ end and a 3′ end; (c) digesting the double stranded nucleic acid comprising one or more uracils at the 5′ end and the 3′ end using a glycosylase and a glycosylase-lyase to generate a double stranded nucleic acid comprising a loop structure at the 5′ end and the 3′ end; and (d) ligating gaps in the double stranded nucleic acid comprising the loop structure at the 5′ end and the 3′ end using a ligase to generate the covalently closed double stranded nucleic acid, wherein the primer comprises a nucleic acid sequence according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

2 . The method of claim 1 , wherein the double stranded nucleic acid is deoxyribonucleic acid.

3 . The method of claim 1 , wherein the double stranded nucleic acid is linear.

4 . The method of claim 1 , wherein the glycosylase comprises base excision activity.

5 . The method of claim 4 , wherein the base excision activity of the glycosylase generates an abasic site.

6 . The method of claim 1 , wherein the glycosylase excises the one or more uracils.

7 . The method of claim 1 , wherein the glycosylase is AlkA, 3-methyladenine DNA glycosylase II, Mag1, MPG, SMUG1, MBD4, NTHL1, uracil DNA glycosylases, helix-hairpin-helix (HhH) glycosylases, or 3-methyl-purine glycosylase (MPG).

8 . The method of claim 5 , wherein the glycosylase-lyase breaks the phosphodiester backbone at a 3′ and 5′ sides of the abasic site.

9 . The method of claim 1 , wherein the glycosylase-lyase is Endonuclease VIII.

10 . The method of claim 1 , wherein the loop structure comprises at most about 40 bases.

11 . The method of claim 1 , wherein the loop structure comprises a sequence according to any one of SEQ ID NOs: 9-20.

12 . The method of claim 1 , wherein step (c) comprises excision of the one or more uracils.

13 . The method of claim 1 , wherein the method does not require heating between step (c) and step (d).

14 . A covalently closed double stranded nucleic acid generated by the method of claim 1 .

15 . A method for assembly of a covalently closed double stranded nucleic acid, comprising:
(a) providing a double stranded nucleic acid; (b) amplifying the double stranded nucleic acid using a primer comprising one or more uracils to generate a double stranded nucleic acid comprising one or more uracils at a 5′ end and a 3′ end; (c) digesting the double stranded nucleic acid comprising one or more uracils at the 5′ end and the 3′ end using a glycosylase and a glycosylase-lyase to generate a double stranded nucleic acid comprising a loop structure at the 5′ end and the 3′ end; and (d) ligating gaps in the double stranded nucleic acid comprising the loop structure at the 5′ end and the 3′ end using a ligase to generate the covalently closed double stranded nucleic acid, wherein the loop structure comprises a nucleic acid sequence according to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

16 . The method of claim 15 , wherein the double stranded nucleic acid is deoxyribonucleic acid.

17 . The method of claim 15 , wherein the double stranded nucleic acid is linear.

18 . The method of claim 15 , wherein the glycosylase comprises base excision activity.

19 . The method of claim 18 , wherein the base excision activity of the glycosylase generates an abasic site.

20 . The method of claim 15 , wherein the glycosylase excises the one or more uracils.

21 . The method of claim 15 , wherein the glycosylase is AlkA, 3-methyladenine DNA glycosylase II, Mag1, MPG, SMUG1, MBD4, NTHL1, uracil DNA glycosylases, helix-hairpin-helix (HhH) glycosylases, or 3-methyl-purine glycosylase (MPG).

22 . The method of claim 19 , wherein the glycosylase-lyase breaks the phosphodiester backbone at a 3′ and 5′ sides of the abasic site.

23 . The method of claim 15 , wherein the glycosylase-lyase is Endonuclease VIII.

24 . The method of claim 15 , wherein the loop structure comprises at most about 40 bases.

25 . The method of claim 15 , wherein step (c) comprises excision of the one or more uracils.

26 . The method of claim 15 , wherein the method does not require heating between step (c) and step (d).

27 . A covalently closed double stranded nucleic acid generated by the method of claim 15 .

Assignees

• Twist Bioscience Corp

Inventors

• Nugent Rebecca
• Chen Siyuan
• Raynard Nathan

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N2310/532

  Closed or circular · CPC title

• C12N15/111

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