Single-stranded splint strands and methods of use

Patent metadata

Field	Value
Publication number	US-2026071207-A1
Application number	US-202519240104-A
Country	US
Kind code	A1
Filing date	Jun 17, 2025
Priority date	Mar 4, 2022
Publication date	Mar 12, 2026
Grant date	—

Abstract

The present disclosure provides compositions comprising nucleic acid single-stranded splint strands, including kits, and methods that employ the single-stranded splint strands. The single-stranded splint strands can hybridize to portions of linear library molecules to form circularized library-splint complexes having a nick, where the nick can be ligated to form covalently closed circular molecules which can be subjected to downstream amplification and sequencing workflows.

First claim

1 .- 28 . (canceled)

29 . A method for generating a plurality of covalently closed circular library molecules ( 400 ) comprising:
a) providing a plurality of single-stranded nucleic acid library molecules ( 100 ), wherein individual library molecules in the plurality comprise regions arranged in a 5′ to 3′ order: (i) a first left universal adaptor sequence ( 120 ); (ii) a second left universal adaptor sequence ( 140 ); (iii) a sequence of interest ( 110 ); (iv) a second right universal adaptor sequence ( 150 ); and (v) a first right universal adaptor sequence ( 130 ), wherein the single-stranded nucleic acid library molecule comprises a first left unique identification sequence ( 180 ) and/or a first right unique identification sequence ( 190 ); b) providing a plurality of single-stranded splint strands ( 200 ), wherein individual single-stranded splint strands ( 200 ) in the plurality comprise a first region ( 210 ) that hybridizes with the first left universal adaptor sequence ( 120 ) of individual single-stranded nucleic acid library molecules, and a second region ( 220 ) that hybridizes with the first right universal adaptor sequence ( 130 ) of individual single-stranded nucleic acid library molecules; c) hybridizing the plurality of single-stranded splint strands ( 200 ) with the plurality of single-stranded nucleic acid library molecules ( 100 ), wherein the first region of one of the single-stranded splint strands ( 210 ) anneals to the first left universal adaptor sequence ( 120 ) of the individual single-stranded nucleic acid library molecule and the second region of the single-stranded splint strand ( 220 ) anneals to the first right universal sequence ( 130 ) of the individual single-stranded nucleic acid library molecule, thereby circularizing the individual single-stranded nucleic acid library molecules and forming a plurality of library-splint complexes ( 300 ) having a nick between the 5′ and 3′ ends of the individual single-stranded nucleic acid library molecule; and d) ligating the nick in the plurality of library-splint complexes ( 300 ), thereby generating a plurality of covalently closed circular library molecules ( 400 ).

30 . The method of claim 29 , further comprising:
(e) distributing the plurality of covalently closed circular library molecules ( 400 ) onto a support having a plurality of surface primers immobilized on the support and hybridizing individual covalently closed circular library molecules ( 400 ) to individual immobilized surface primers, thereby immobilizing the plurality of covalently closed circular library molecules ( 400 ) to the support.

31 . The method of claim 30 , further comprising:
(f) contacting the plurality of covalently closed circular library molecules ( 400 ) 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 surface primers as immobilized amplification primers and the plurality of covalently closed circular library molecules ( 400 ) as template molecules, thereby generating a plurality of immobilized nucleic acid concatemer molecules.

32 . The method of claim 31 , further comprising sequencing the plurality of immobilized nucleic acid concatemer molecules.

33 . The method of claim 32 , 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 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 an immobilized nucleic acid concatemer molecule hybridized to a soluble sequencing primer; b) contacting the plurality of complexed polymerases with a plurality of nucleotides and binding at least one nucleotide to a complexed 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 the at least one nucleotide into a 3′ end of a hybridized soluble sequencing primer, thereby generating a plurality of nascent extended sequencing primers; and d) detecting the incorporated nucleotide and identifying the nucleo-base of the incorporated nucleotide.

34 . The method of claim 32 , 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 soluble sequencing primers thereby forming a plurality of first complexed polymerases comprising a sequencing polymerase bound to a nucleic acid duplex, wherein the nucleic acid duplex comprises an immobilized nucleic acid concatemer molecule hybridized to a soluble sequencing primer; b) contacting the plurality of first complexed polymerases with a plurality of detectably labeled multivalent molecules and binding complementary nucleotide units of individual detectably labeled multivalent molecules to at least two first complexed polymerases, thereby forming a plurality of multivalent-complexed polymerases, wherein incorporation of the complementary nucleotide units into the soluble sequencing primers of the plurality of multivalent-complexed polymerases is inhibited, wherein the individual detectably labeled multivalent molecules comprise a core attached to multiple nucleotide arms attached to a nucleotide unit; c) detecting the plurality of multivalent-complexed polymerases; and d) identifying the nucleo-base of the complementary nucleotide units that are bound to the plurality of first complexed polymerases in the plurality of multivalent-complexed polymerases, thereby determining the sequence of the nucleic acid concatemer template molecules.

35 . The method of claim 34 , further comprising:
e) dissociating the plurality of multivalent-complexed polymerases and removing the plurality of first complexed polymerases and the detectably labeled multivalent molecules, and retaining the plurality of nucleic acid duplexes; f) contacting the plurality of nucleic acid duplexes of step (e) with a plurality of second sequencing polymerases and binding the plurality of second sequencing polymerases to the plurality of the nucleic acid duplexes, thereby forming a plurality of second complexed polymerases comprising a second sequencing polymerase bound to a nucleic acid duplex; and g) contacting the plurality of second complexed polymerases with a plurality of nucleotides and binding complementary nucleotides from the plurality of nucleotides to at least two second complexed polymerases of step (f), thereby forming a plurality of nucleotide-complexed polymerases and incorporating the bound complementary nucleotides into the soluble sequencing primers of the nucleic acid duplex.

36 . The method of claim 35 , further comprising:
h) detecting the complementary nucleotides which are incorporated into the sequencing primers of the nucleotide-complexed polymerases; and i) identifying the nucleo-bases of the complementary nucleotides which are incorporated into the sequencing primers of the nucleotide-complexed polymerases.

37 . The method of claim 35 , wherein the plurality of nucleotides comprise a plurality of non-labeled nucleotides.

38 . The method of claim 29 , wherein individual single-stranded nucleic acid library molecules ( 100 ) comprise:
(vi) a first left sample index sequence ( 160 ), and/or (vii) a first right sample index sequence ( 170 ).

39 . The method of claim 29 ,
(A) wherein the first left universal adaptor sequence ( 1

Assignees

• Element Biosciences Inc

Inventors

• Light William
• Kelley Ryan
• Zhao Junhua

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6853

  using modified primers or templates · 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

• C12Q2535/125

  Allele specific primer extension · CPC title

• C12Q2525/191

  incorporating an adaptor · CPC title