IP Library › Granted Patent US 12,540,321
Granted Patent B2
US 12,540,321 · App. 18/299,553 · Granted Feb 3, 2026

Methods of producing nucleic acid libraries and compositions and kits for practicing same

Inventors: Richard Green (Santa Cruz, CA); Joshua Kapp (Santa Cruz, CA)
Assignee: The Regents of the University of California
C12N15/1093C12Q1/6869
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Quick Facts
Patent No.
US 12,540,321
App. No.
18/299,553
Filed
Apr 12, 2023
Granted
Feb 3, 2026
Kind
B2
Art Unit
1684
USPC
506/2
Abstract

Provided are methods of producing nucleic acid libraries. The methods include combining single-stranded nucleic acid binding protein-bound single-stranded nucleic acid (SSB-bound ssNA), an adapter oligonucleotide, and a splint oligonucleotide, to form complexes including the splint oligonucleotide hybridized to a terminal region of the SSB-bound ssNA and to the adapter oligonucleotide. An end of the first adapter oligonucleotide is adjacent to an end of the first terminal region of the SSB-bound ssNA, and the methods may further include covalently linking the adjacent ends. Also provided are compositions and kits that find use, e.g., in practicing the methods of the present disclosure.

Claims (24)

1 . A composition, comprising:

a complex comprising:

single-stranded nucleic acid binding protein-bound single-stranded nucleic acid (SSB-bound ssNA);

a first adapter oligonucleotide;

a first splint oligonucleotide comprising an SSB-bound ssNA hybridization region and a first adapter oligonucleotide hybridization region, wherein the first adapter oligonucleotide and the first adapter oligonucleotide hybridization region are the same length;

a second adapter oligonucleotide; and

a second splint oligonucleotide comprising an SSB-bound ssNA hybridization region and a second adapter oligonucleotide hybridization region, wherein the second adapter oligonucleotide and the second adapter oligonucleotide hybridization region are the same length,

wherein the complex comprises:

the first splint oligonucleotide hybridized to a terminal region of the SSB-bound ssNA via the SSB-bound ssNA hybridization region, and the first splint oligonucleotide hybridized to the first adapter oligonucleotide via the first adapter oligonucleotide hybridization region, such that an end of the first adapter oligonucleotide is adjacent to an end of the terminal region of the SSB-bound ssNA, wherein the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a ligation-blocking modification; and

the second splint oligonucleotide hybridized to the terminal region of the SSB-bound ssNA not hybridized to the first splint oligonucleotide via the SSB-bound ssNA hybridization region of the second splint oligonucleotide, and the second splint oligonucleotide hybridized to the second adapter oligonucleotide via the second adapter oligonucleotide hybridization region, such that an end of the second adapter oligonucleotide is adjacent to the end of the terminal region of the SSB-bound ssNA hybridized to the second splint oligonucleotide, wherein the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA hybridized to the second splint oligonucleotide comprises a ligation-blocking modification.

2 . The composition of claim 1 , further comprising a ligase.

3 . The composition of claim 1 , wherein the adjacent ends of the first adapter oligonucleotide and SSB-bound ssNA are covalently linked.

4 . The composition of claim 3 , wherein the adjacent ends of the second adapter oligonucleotide and SSB-bound ssNA hybridized to the second splint oligonucleotide are covalently linked.

5 . The composition of claim 1 , wherein the ligation-blocking modification comprises the absence of a 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA and/or the absence of a 3′ OH at the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA.

6 . The composition of claim 1 , wherein the ligation-blocking modification comprises an inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA and/or an inaccessible 3′ OH at the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA.

7 . The composition of claim 6 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA and/or at the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises an amino modifier.

8 . The composition of claim 6 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA and/or at the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a spacer.

9 . The composition of claim 6 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA and/or at the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a dideoxy base.

10 . The composition of claim 6 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA and/or at the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises an inverted dideoxy base.

11 . The composition of claim 6 , wherein the ligation-blocking modification comprising the inaccessible 3′ OH at the end of the first adapter oligonucleotide not adjacent to the SSB-bound ssNA and/or at the end of the second adapter oligonucleotide not adjacent to the SSB-bound ssNA comprises a 3′ phosphate.

12 . The composition of claim 1 , wherein the ssNA is single-stranded DNA.

13 . The composition of claim 12 , wherein the single-stranded DNA is single-stranded genomic DNA.

14 . The composition of claim 1 , wherein the ssNA is RNA.

15 . The composition of claim 13 , wherein the RNA is messenger RNA (mRNA).

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 13, 2023
From: GREEN, RICHARD; KAPP, JOSHUA
To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Reel/Frame 064236/0853 →
Continuity (3)
Division 17058066
Provisional Application 62681524 · Jun 6, 2018
Related Publication 20230242907A1 · Aug 3, 2023
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