Compositions and methods for preparing nucleic acid nanostructures using compaction oligonucleotides

Patent metadata

Field	Value
Publication number	US-12421545-B2
Application number	US-202318450302-A
Country	US
Kind code	B2
Filing date	Aug 15, 2023
Priority date	Aug 15, 2022
Publication date	Sep 23, 2025
Grant date	Sep 23, 2025

Abstract

The present disclosure provides compositions and related methods, e.g., for preparing immobilized nucleic acid nanostructures using compaction oligonucleotides. In some embodiments, rolling circle amplification reaction can be conducted with compaction oligonucleotides on-support or in-solution to generate concatemer molecules having multiple copies of a polynucleotide unit arranged in tandem. Each polynucleotide unit comprises a sequence-of-interest and at least one universal adaptor sequence that binds one end of a compaction oligonucleotide. The 5′ and 3′ regions of the compaction oligonucleotide can hybridize to the concatemer to pull together distal portions of the concatemer causing compaction of the concatemer to form a nanostructure. Nanostructures having tighter size and shape compared to concatemers generated in the absence of the compaction oligonucleotides. The compact and stable characteristics of the nucleic acid nanostructures improves sequencing accuracy by increasing signal intensity and they retain their shape and size during multiple sequencing cycles.

First claim

What is claimed:

1. A method comprising:
a) providing a support having a plurality of first universal surface primers immobilized thereon, wherein the density of the first universal surface primers on the support is between about 10 2 -10 15 per mm 2 ; and
b) generating a plurality of immobilized single stranded nucleic acid concatemer template molecules, the generating comprising:
1) hybridizing a plurality of single stranded circular nucleic acid library molecules to the plurality of first universal surface primers; and
2) conducting an on-support rolling circle amplification reaction, wherein the reaction comprises contacting the plurality of single stranded circular nucleic acid library molecules with (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides, and (iii) a plurality of compaction oligonucleotides, individual compaction oligonucleotides comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, and 156,

thereby generating the plurality of immobilized single stranded nucleic acid concatemer template molecules,
wherein individual compaction oligonucleotides comprise a single-stranded linear oligonucleotide having a first binding region capable of hybridizing to a first portion of an individual immobilized single stranded nucleic acid concatemer template molecule, and a second binding region capable of hybridizing to a second portion of the individual immobilized single stranded nucleic acid concatemer template molecule,
wherein the plurality of immobilized single stranded nucleic acid concatemer template molecules form compact nucleic acid nanostructures, and
wherein the plurality of immobilized single stranded nucleic acid concatemer template molecules remains immobilized to the support upon forming the compact nucleic acid nanostructures, thereby generating a density of about 10 2 -10 15 per mm 2 of compact nucleic acid nanostructures immobilized on the support.

2. The method of claim 1 , wherein the support is passivated with at least one layer of a hydrophilic polymer coating comprising the plurality of first universal surface primers.

3. The method of claim 1 , wherein the plurality of first universal surface primers is located on the support or a hydrophilic polymer coating at random positions or pre-determined positions.

4. The method of claim 1 , wherein each of the individual first universal surface primers of the plurality lack a scissile moiety that can be converted into abasic sites, and wherein the scissile moiety comprises a uridine, an 8-oxo-7,8-dihydroguanine, or a deoxyinosine.

5. The method of claim 1 , wherein:
a) the plurality of nucleotides for the rolling circle amplification reaction comprises dATP, dCTP, dGTP and dTTP, and wherein the nucleotides lack a scissile moiety that can be converted into an abasic site; or
b) the plurality of nucleotides for the rolling circle amplification reaction comprises dATP, dCTP, dGTP, dTTP, and comprises nucleotides having a scissile moiety that can be converted into abasic sites,
wherein the nucleotides having the scissile moiety comprise uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine.

6. The method of claim 5 , wherein individual immobilized single stranded nucleic acid concatemer template molecules include at least two nucleotides each having a scissile moiety distributed at random positions along the individual immobilized single stranded nucleic acid concatemer template molecules.

7. The method of claim 1 , wherein the plurality of compaction oligonucleotides in step (b):
a) comprises a population of compaction oligonucleotides having the same sequence, or
b) comprises a mixture of two or more different populations of compaction oligonucleotides, each population having different sequences, wherein the compaction oligonucleotides in the different populations have different sequences.

8. The method of claim 1 , wherein the compact nucleic acid nanostructures comprise one or more loops, or comprise a spherical shape, elongated shape, proto-toroid shape, or toroid shape, wherein:
a) the spherical shape is a nanoball;
b) the elongated shape is a nanorod, or
c) the toroid shape is a nano-toroid.

9. The method of claim 1 , wherein the compact nucleic acid nanostructures comprise a full width half maximum (FWHM) smaller than an immobilized single stranded nucleic acid concatemer template molecule that is not collapsed/folded into a nanostructure.

10. The method of claim 1 , further comprising imaging the compact nucleic acid nanostructures immobilized on the support.

11. The method of claim 1 , further comprising:
1) A) contacting the plurality of compact nucleic acid nanostructures with labeled oligonucleotides comprising detectable reporter moieties under a condition suitable for hybridizing the labeled oligonucleotides to the compact nucleic acid nanostructures to generate a plurality of labeled nanostructures; and
b) imaging the plurality of labeled nanostructures, or

2) Contacting the plurality of compact nucleic acid nanostructures with (i) a plurality of soluble sequencing primers, (ii) a plurality of sequencing polymerases, and (iii) a plurality of nucleotide reagents, under a condition suitable for:
hybridizing the plurality of soluble sequencing primers to individual compact nucleic acid nanostructures to generate a plurality of nucleic acid duplexes along individual compact nucleic acid nanostructures, and
binding at least one nucleic acid duplex with a sequencing polymerase and a nucleotide reagent,

wherein the plurality of nucleotide reagents comprises a plurality of nucleotides, wherein individual nucleotides comprise an aromatic base, a five-carbon sugar, and at least one phosphate group.

12. The method of claim 11 , wherein at least one of the nucleotides in the plurality further comprises a detectable reporter moiety, wherein the detectable reporter moiety is a fluorophore.

13. The method of claim 12 , further comprising:
a) contacting the plurality of compact nucleic acid nanostructures with a labeled nucleotide; and
b) imaging the plurality of compact nucleic acid nanostructures immobilized on the support.

14. The method of claim 11 , wherein the plurality of nucleotide reagents comprises a plurality of nucleotide analogs, wherein individual nucleotide analogs comprise an aromatic base, a five-carbon sugar having a 3′ chain terminating moiety that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group, and
wherein at least one of the nucleotide analogs in the plurality further comprises a detectable reporter moiety, wherein the detectable reporter moiety is a fluorophore.

15. The method of claim 14 , further comprising:
a) contacting the plurality of compact nucleic acid nanostructures with a labeled nucleotide analog; and
b) imaging the plurality of compact nucleic acid nanostructures immobilized on the support.

16. The method of claim 11 , wherein the plurality of nucleotide reagents comprises a plurality of multivalent molecules, wherein individual multivalent molecules comprise: (1) a core; and (2) a plurality of nucleotide arms which comprise (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, the spacer is attached to the linker, the linker is attached to the nucl

Assignees

• Element Biosciences Inc

Inventors

• Arslan Sinan
• Kim Michael
• Tippana Ramreddy
• Zhou Chunhong
• Light William
• Yu Hua
• Zhao Junhua
• Liu Tsung-Li

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2565/537

  characterised by the capture oligonucleotide acting as a primer · CPC title

• C12Q2565/519

  characterised by the capture moiety being a single stranded oligonucleotide · CPC title

• C12Q2565/507

  characterised by the density of the capture oligonucleotide · CPC title