Double-stranded splint adaptors and methods of use

Patent metadata

Field	Value
Publication number	US-2026071266-A1
Application number	US-202519244055-A
Country	US
Kind code	A1
Filing date	Jun 20, 2025
Priority date	Mar 4, 2022
Publication date	Mar 12, 2026
Grant date	—

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

1 .- 28 . (canceled)

29 . A library-splint complex ( 500 ) comprising:
(i) a single-stranded nucleic acid library molecule ( 100 ) comprising a sequence of interest ( 110 ) flanked on one side by at least a first left universal adaptor sequence ( 120 ) and flanked on the other side by at least a first right universal adaptor sequence ( 130 ); and (ii) a double-stranded splint adaptor ( 200 ) comprising a first splint strand ( 300 ) and a second splint strand ( 400 ), wherein the double-stranded splint adaptor ( 200 ) comprises a double-stranded region and two flanking single-stranded regions, wherein the first splint strand 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 ); wherein the internal region of the first splint strand ( 310 ) is hybridized to the second splint strand ( 400 ), wherein the first region of the first splint strand ( 320 ) is hybridized to the at least first left universal adaptor sequence ( 120 ) of the library molecule, and wherein the second region of the first splint strand ( 330 ) is hybridized to the at least first right universal sequence ( 130 ) of the library molecule, thereby circularizing the library molecule to generate a library-splint complex ( 500 ).

30 . The library-splint complex ( 500 ) of claim 29 , wherein the single-stranded nucleic acid library molecule ( 100 ) comprises any one or any combination of two or more of:
(i) a second left universal adaptor sequence ( 140 ); (ii) a second right universal adaptor sequence ( 150 ); (iii) a first left index sequence ( 160 ); (iv) a first right index sequence ( 170 ); (v) a first left unique identification sequence ( 180 ); and/or (vi) a first right unique identification sequence ( 190 ).

31 . The library-splint complex ( 500 ) of claim 29 , wherein:
a) the first left universal adaptor sequence ( 120 ) and/or the second left universal adaptor sequence ( 140 ) comprises: (i) a universal binding sequence for a forward sequencing primer; (ii) a universal binding sequence for a reverse sequencing primer; (iii) a universal binding sequence for a first surface primer; (iv) a universal binding sequence for a second surface primer; (v) a universal binding sequence for a forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and/or (vii) a universal binding sequence for a compaction oligonucleotide; or b) the first right universal adaptor sequence ( 130 ) and/or the second right universal adaptor sequence ( 150 ) comprises: (i) a universal binding sequence for a forward sequencing primer; (ii) a universal binding sequence for a reverse sequencing primer; (iii) a universal binding sequence for a first surface primer; (iv) a universal binding sequence for a second surface primer; (v) a universal binding sequence for a forward amplification primer; (vi) a universal binding sequence for a reverse amplification primer; and/or (vii) a universal binding sequence for a compaction oligonucleotide.

32 . The library-splint complex ( 500 ) of claim 29 , wherein the second splint strand ( 400 ) includes at least two sub-regions comprising a first sub-region comprising a universal binding sequence for a third surface primer, and a second sub-region comprising a universal binding sequence for a fourth surface primer, wherein the first and the second sub-regions do not hybridize to or exhibit very little hybridization to the first and the second surface primers.

33 . The library-splint complex ( 500 ) of claim 32 , wherein the second splint strand ( 400 ) comprises a third sub-region comprising a sample index sequence having 5-20 bases, a unique identification sequence having 2-10 or more bases, or a combination thereof.

34 . The library-splint complex ( 500 ) of claim 32 , wherein the first splint strand ( 300 ) comprises an internal region ( 310 ) comprising at least two sub-regions comprising (i) a fourth sub-region comprising a universal binding sequence for a third surface primer, wherein the fourth sub-region hybridizes to the first sub-region of the second splint strand ( 400 ), and (ii) a fifth sub-region comprising a universal binding sequence for a fourth surface primer, wherein the fifth sub-region hybridizes to the second sub-region of the second splint strand ( 400 ), and wherein the fourth and fifth sub-regions do not hybridize to or exhibit very little hybridization to the first and second surface primers.

35 . The library-splint complex ( 500 ) of claim 34 , wherein the internal region ( 310 ) of the first splint strand comprises a sixth sub-region comprising a sample index sequence having 5-20 bases, a unique identification sequence having 2-10 or more bases, or a combination thereof, wherein the sixth sub-region hybridizes to the third sub-region of the second splint strand ( 400 ).

36 . The library-splint complex ( 500 ) of claim 29 , comprising:
a) a first nick between a 5′ end of the single-stranded nucleic acid library molecule and a 3′ end of the second splint strand, and/or b) a second nick between a 5′ end of the second splint strand and a 3′ end of the single-stranded nucleic acid library molecule, wherein the first nick and/or the second nick are enzymatically ligatable.

37 . A method for forming a plurality of library-splint complexes ( 500 ) comprising:
a) providing a plurality of double-stranded splint adaptors, wherein individual double-stranded splint adaptors ( 200 ) comprise a first splint strand ( 300 ) hybridized to a second splint strand ( 400 ), wherein individual double-stranded splint adaptors include a double-stranded region and two flanking single-stranded regions, wherein the first splint strand 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 ); and b) hybridizing the plurality of double-stranded splint adaptors with a plurality of single-stranded nucleic acid library molecules ( 100 ), wherein individual single-stranded nucleic acid library molecules include a sequence of interest ( 110 ) flanked on one side by at least a first left universal adaptor sequence ( 120 ) and flanked on the other side by at least a first right universal adaptor sequence ( 130 ), wherein the hybridizing is conducted under a condition suitable to hybridize the first region of the first splint strand ( 320 ) to the at least first left universal adaptor sequence ( 120 ) of the single-stranded nucleic acid library molecule, and the condition is suitable to hybridize the second region of the first splint strand ( 330 ) to the at least first right universal sequence ( 130 ) of the single-stranded nucleic acid library molecule, thereby circularizing the plurality of single-stranded nucleic acid library molecules to form a plurality of library-splint complexes ( 500 ).

38 . The method of claim 37 , wherein individual library-splint complexes ( 500 ) in the plurality comprise a first nick between a 5′ end of the library molecule and a 3′ end of the second splint strand, and a second nick between a 5′ end of the second splint strand and a 3′ end of the library molecule.

39 . The method of claim 37 , wherein individual single-stranded nucleic acid library molecules ( 100 ) in the plurality comprise any one or any combination of two or more of:
(i) a second left universal adaptor sequence ( 140 ); (ii) a second right universal adaptor sequence ( 150 ); (iii) a first left index sequence ( 160 ); (iv) a first right index

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