Double-stranded splint adaptors and methods of use

Patent metadata

Field	Value
Publication number	US-12371743-B2
Application number	US-202217725065-A
Country	US
Kind code	B2
Filing date	Apr 20, 2022
Priority date	Mar 4, 2022
Publication date	Jul 29, 2025
Grant date	Jul 29, 2025

Abstract

The present disclosure provides compositions comprising nucleic acid double-stranded splint adaptors, including kits, and methods that employ the double-stranded splint adaptors. The double-stranded splint adaptors ( 200 ) can be used in a one-pot, multi-enzyme reaction to introduce one or more new adaptor sequences into a library molecule. The double-stranded splint adaptor ( 200 ) comprises a first splint strand (long splint strand ( 300 )) and a second splint strand (short splint strand ( 400 )), where the first and second splint strands are hybridized together to form the double-stranded splint adaptor ( 200 ) having a double-stranded region and two flanking single-stranded regions. The second splint strand ( 400 ) carries the new adaptor sequence(s) to be introduced, such as for example a universal binding sequence and/or an index sequence.

First claim

What is claimed:

1. A method for forming a plurality of library-splint complexes ( 500 ) on a support, the method comprising:
a) providing a plurality of double-stranded splint adaptors ( 200 ), wherein individual double-stranded splint adaptors ( 200 ) comprise a first splint strand ( 300 ) hybridized to a second splint strand ( 400 ), wherein the first splint strand ( 300 ) comprises regions arranged in a 5′ to 3′ order: (i) a first region ( 320 ), (ii) an internal region ( 310 ), and (iii) a second region ( 330 ), and wherein the internal region of the first splint strand ( 310 ) is hybridized to the second splint strand ( 400 ), wherein the second splint strand comprises regions arranged in a 5′ to 3′ order: (i) a second sub-region having a universal binding sequence for a first surface primer, and (ii) a first sub-region having a universal binding sequence for a second surface primer; and
b) hybridizing the plurality of double-stranded splint adaptors with a plurality of single-stranded nucleic acid library molecules ( 100 ), wherein individual library molecules comprise regions arranged in a 5′ to 3′ order: (i) a first left universal adaptor sequence ( 120 ) having a binding sequence for a third surface primer; (ii) a second left universal adaptor sequence ( 140 ) having a binding sequence for a first sequencing primer; (iii) a sequence of interest ( 110 ); (iv) a second right universal adaptor sequence ( 150 ) having a binding sequence for a second sequencing primer; and (v) a first right universal adaptor sequence ( 130 ) having a binding sequence for a fourth surface primer,
wherein portions of the first splint strand ( 300 ) hybridize to portions of the library molecule ( 100 ) thereby circularizing the library molecule to generate a library-splint complex ( 500 ), such that the first region ( 320 ) of the first splint strand is hybridized to the binding sequence for the third surface primer ( 120 ), and the second region ( 330 ) of the first splint strand is hybridized to the binding sequence for the fourth surface primer ( 130 ), wherein the library-splint complex ( 500 ) comprises a first nick between the 5′ end of the library molecule and the 3′ end of the second splint strand ( 400 ), wherein the library-splint complex ( 500 ) comprises a second nick between the 5′ end of the second splint strand ( 400 ) and the 3′ end of the library molecule ( 100 ), and wherein the first and second nicks are enzymatically ligated to generate a plurality of covalently closed circular library molecules ( 600 ); and

c) distributing the plurality of covalently closed circular library molecules ( 600 ) onto a support having a plurality of the second surface primers and first surface primers immobilized on the support, wherein individual covalently closed circular library molecules ( 600 ) hybridize to individual second surface primers and first surface primers thereby immobilizing the plurality of covalently closed circular library molecules ( 600 ) to the support,

and wherein the plurality of second surface primers and first surface primers on the support are located at pre-determined or random locations on the support.

2. The method of claim 1 , wherein the plurality of single-stranded nucleic acid library molecules ( 100 ) further comprises a first left index sequence ( 160 ) and/or a first right index sequence ( 170 ).

3. The method of claim 1 , where wherein the plurality of single-stranded nucleic acid library molecules ( 100 ) further comprises a first left unique identification sequence ( 180 ) and/or a first right unique identification sequence ( 190 ).

4. The method of claim 1 , wherein the plurality of covalently closed circular library molecules ( 600 ) of b) are each hybridized to the first splint strand ( 300 ).

5. The method of claim 4 , further comprising: contacting the plurality of covalently closed circular library molecules ( 600 ) of b) with at least one exonuclease enzyme to remove the plurality of first splint strands ( 300 ) and retaining the plurality of covalently closed circular library molecules ( 600 ).

6. The method of claim 1 , further comprising: d) contacting the plurality of covalently closed circular library molecules ( 600 ) immobilized on the support with a plurality of strand-displacing polymerases and a plurality of nucleotides, and conducting a rolling circle amplification reaction on the support using the plurality of second surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules ( 600 ) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules.

7. The method of claim 6 , wherein the plurality of nucleotides comprises dATP, dGTP, dCTP, dTTP and/or dUTP.

8. The method of claim 6 , wherein the plurality of second surface primers and first surface primers on the support are in fluid communication with each other to permit flowing a solution of reagents onto the support so that the plurality of second surface primers can be simultaneously reacted with the reagents in a massively parallel manner, optionally wherein the solution of reagents comprises enzymes, nucleotides and divalent cations.

9. The method of claim 6 , wherein the density of the plurality of immobilized nucleic acid concatemer molecules on the support is 10 4 -10 8 per mm 2 .

10. The method of claim 6 , further comprising: sequencing the plurality of immobilized nucleic acid concatemer molecules, wherein the sequencing comprises:
a) contacting the plurality of immobilized nucleic acid concatemer molecules with (i) a plurality of sequencing polymerases and (ii) a plurality of the soluble sequencing primers, thereby forming a plurality of complexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises a concatemer molecule hybridized to a soluble sequencing primer;
b) contacting the plurality of complexed sequencing polymerases with a plurality of nucleotides and binding at least one nucleotide to a complexed sequencing polymerase, wherein the plurality of nucleotides comprises at least one nucleotide analog labeled with a fluorophore and having a removable chain terminating moiety at the sugar 3′ position;
c) incorporating at least one nucleotide into the 3′ end of the hybridized sequencing primers thereby generating a plurality of nascent extended sequencing primers; and
d) detecting the incorporated nucleotide and identifying the nucleo-base of the incorporated nucleotide.

11. The method of claim 10 , wherein the plurality of nucleotides comprises a removable chain terminating moiety at the 3′ sugar group, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, or silyl group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3′OH moiety on the sugar group.

12. The method of claim 10 , wherein the plurality of nucleotides comprises one type of nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

13. The method of claim 10 , wherein the plurality of nucleotides comprises a mixture of any combination of two or more types of nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

14. The method of claim 6 , further comprising: sequencing the plurality of immobilized nucleic acid concatemer molecul

Assignees

• Element Biosciences Inc

Inventors

• Light William
• Snow Samantha
• Zhao Junhua

Classifications

• C12Q1/6855 Primary

  Ligating adaptors · CPC title

• C12Q1/682

  Signal amplification · CPC title

• C12N15/1068

  Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q2535/125

  Allele specific primer extension · CPC title