Controlled polynucleotide translocation in nanopore sequencing

Patent metadata

Field	Value
Publication number	US-2025019758-A1
Application number	US-202418650800-A
Country	US
Kind code	A1
Filing date	Apr 30, 2024
Priority date	May 3, 2023
Publication date	Jan 16, 2025
Grant date	—

Abstract

Provided herein are methods for modification-based controlled polynucleotide translocation in nanopores for sequencing, modified nucleotides, and kits and systems for performing the disclosed methods. In some embodiments, modifications can be used to control polynucleotide translocation by modifying nucleotides on a strand of polynucleotide to carry a modification, where the modifications can arrest or slow translocation when encountering the nanopore. In some embodiments, application of a voltage can move one nucleotide and its attached modification through the nanopore at a time.

First claim

What is claimed is:

1 . A method for determining a sequence of a target polynucleotide with a nanopore-based sequencing system, the method comprising:
providing a target polynucleotide comprising nucleotides, wherein each nucleotide comprises a modification, wherein the modification comprises an arresting construct configured to arrest translocation of the target polynucleotide through a nanopore; applying a driving voltage to translocate one or more portions of the target polynucleotide through a nanopore; measuring a current of the nanopore continuously during translocation; and identifying the sequence of the target polynucleotide by correlating the measured current to an identity of one or more nucleotides.

2 . The method of claim 1 , wherein providing the target polynucleotide comprises synthesizing a daughter strand based on a template polynucleotide using nucleotides with modifications, wherein the modifications are covalently attached to the nucleotides.

3 . The method of any one of claim 1 , wherein the modification further comprises a cyclic loop, wherein the arresting construct is attached to the cyclic loop.

4 . The method of claim 3 , wherein the cyclic loop further comprises a reporter element that encodes the nucleotide.

5 . The method of claim 4 , wherein the cyclic loop further comprises a spacer.

6 . The method of claim 1 , wherein providing the target polynucleotide comprises:
synthesizing a daughter strand based on a template polynucleotide using nucleotides with modifications, wherein the modifications are covalently attached to the nucleotides; and cleaving the daughter strand to generate the target polynucleotide having an elongated polynucleotide strand.

7 . The method of claim 1 , wherein the nucleotide has a dwell time in the nanopore of greater than 5 ms.

8 . The method of claim 1 , wherein the nucleotide has a dwell time in the nanopore of greater than 0.1 ms.

9 . The method of claim 1 , wherein the driving voltage is kept constant during the translocation.

10 . The method of claim 4 , wherein the measured current is dependent on the reporter element or the nucleotide passing through the nanopore.

11 . The method of claim 1 , wherein the arresting construct comprises a polymer selected from the group consisting of a linear synthetic hydrophilic polymer, linear synthetic hydrophobic polymer, linear polynucleotide, linear polypeptide, branched polymer, dendritic polymer, cyclic polymer, fluoroalkyl, rigid conjugated chromophores, and rigid macrocycles.

12 . The method of claim 11 , wherein the arresting construct comprises a covalent coupling between the polymer and the corresponding nucleotide or a cyclic loop.

13 . The method of claim 12 , wherein the covalent coupling is selected from the group consisting of amine-NHS ester, amine-imidoester, amine-pentofluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azide-norbornene.

14 . The method of claim 11 , wherein the linear synthetic hydrophilic polymer is selected from the group consisting of polyethyleneglycol, polyvinylalcohol, polyacrylamide, polyvinylpyrrolidone, polystyrenesulfonate, polyethyleneimine, and a combination thereof.

15 . The method of claim 11 , wherein the linear synthetic hydrophobic polymer is selected from the group consisting of polylactic acid, polymethymethacrylate, polystyrene, and a combination thereof.

16 . The method of claim 11 , wherein the linear polynucleotide is a homopolymer of a natural nucleotide, a homopolymer of an unnatural nucleotide, a mixed sequence polymer of natural nucleotides, or a mixed sequence polymer of unnatural nucleotides.

17 . The method of claims 11 , wherein the linear polypeptide comprises one or more types of amino acids.

18 . The method of claim 11 , wherein the branched polymer comprises 2 or more branches.

19 . The method of any one of claims 11 , wherein the cyclic polymer has 3 or more repeating units.

20 . The method of claim 19 , each repeating unit is a small molecule, a nucleotide, or an amino acid.

21 . The method of claim 19 , wherein the repeating units are the same.

22 . The method of claim 19 , wherein at least two of the repeating units are different.

23 . The method of claim 1 , wherein the arresting construct interacts with the nanopore via a non-covalent interaction.

24 . The method of claim 23 , wherein the non-covalent interaction comprises electrostatic interactions, ion-dipole interactions, dipole-dipole interactions, hydrophobic interactions, and combinations thereof.

25 . A system for determining a sequence of a target polynucleotide using a method according to claim 1 .

26 . A cyclic loop nucleotide comprising a cyclic loop modification bridging a nucleobase and a phosphate group, wherein the cyclic loop modification comprises a reporter encoding the identity of the nucleobase, and an arresting construct adjacent to the reporter.

27 . The cyclic loop nucleotide of claim 26 , wherein the arresting construct is adjacent to the reporter.

28 . The cyclic loop nucleotide of claim 26 , wherein the arresting construct comprises a linear, branched, cyclic, or dendritic structure.

29 . The cyclic loop nucleotide of claim 26 , wherein the cyclic loop nucleotide has one of the following structures:

wherein:
X is —O—, —CH 2 —, —NSO 2 —, —NH—,

X′ is —S—, ═N—SO 2 —; ═NH—CO—, or

Base is the nucleobase;
L 1 and L 2 are each a linking group;
RP is the reporter encoding the nucleobase;
and ARC is the arresting construct.

30 . The cyclic loop nucleotide of claim 29 , wherein the ARC is covalently attached to the cyclic loop via a covalent coupling selected from amine-NHS ester, amine-imidoester, amine-pentofluorophenyl ester, amine-hydroxymethyl phosphine, carboxyl-carbodiimide, thiol-maleimide, thiol-haloacetyl, thiol-pyridyl disulfide, thiol-thiosulfonate, thiol-vinyl sulfone, aldehyde-hydrazide, aldehyde-alkoxyamine, hydroxy-isocyanate, azide-alkyne, azide-phosphine, transcyclooctene-tetrazine, norbornene-tetrazine, azide-cyclooctyne, and azide-norbornene.

31 . A kit for performing a method for determining a sequence of a target polynucleotide in a nanopore-based sequencing system, the kit comprising one or more cyclic loop nucleotides according to claim 26 .

Assignees

• Illumina Inc

Inventors

• Salam Abdul Sadeer Abd
• Yang Xiangyuan
• Bohra Hassan Zakiruddin
• Teo Yin Nah
• Yuen Tsz Ying
• Lukamto Daniel Hartoyo
• Garcia Erin
• Mandell Jeffrey G

Classifications

• C12Q1/6806 Primary

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

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2525/117

  incorporating modified base · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title