Granted Patent B2

US 12,571,024 · App. 17/820,856 · Granted Mar 10, 2026

Inventors: Rebecca Nugent (San Francisco, CA); Siyuan Chen (San Mateo, CA); Nathan Raynard (San Mateo, CA)

Assignee: Twist Bioscience Corporation

C12Q1/6806C12N15/1093C12P19/34

Abstract

Claims (37)

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 .

Assignments (1)

Continuity (2)

References Cited (330)