Modified nucleotides and related methods for nanopore sequencing

Patent metadata

Field	Value
Publication number	US-2026055452-A1
Application number	US-202519305635-A
Country	US
Kind code	A1
Filing date	Aug 20, 2025
Priority date	Aug 21, 2024
Publication date	Feb 26, 2026
Grant date	—

Abstract

Disclosed herein is a modified oligonucleotide, the modified oligonucleotide comprises one or more modifications in at least one of a phosphate backbone, a nucleobase, or a sugar. The one or more modifications impede translocation through a nanopore by increasing non-covalent interactions with the interior of the nanopore or the lipid bilayer that supports the nanopore, increasing the bulk size or the steric hinderance of oligonucleotide, or alter the charge density of the oligonucleotide.

First claim

What is claimed is:

1 . A modified oligonucleotide comprising one or more modifications in at least one of a phosphate backbone, a nucleobase, or a sugar, wherein the one or more modifications impede translocation through a nanopore.

2 . The modified oligonucleotide of claim 1 , wherein the one or more modifications are synthetic moieties to:
a) increase non-covalent interactions with an interior of the nanopore; b) increase the bulk size of the oligonucleotide; c) alter the charge density of the oligonucleotide; d) increase the steric hinderance of the oligonucleotide; and/or e) increase non-covalent interactions with a lipid bilayer that supports the nanopore.

3 . The modified oligonucleotide of claim 1 , wherein the one or more modifications replaces a non-bridging oxygen on the phosphate backbone and is selected from the group consisting of:

4 . The modified oligonucleotide of claim 1 , wherein the one or more modifications introduces chirality to a non-bridging oxygen on the phosphate backbone.

5 . The modified oligonucleotide of claim 1 , wherein the one or more modifications replaces a single bridging oxygen in the phosphate backbone with one or more elements selected from O, S, and —NH—.

6 . The modified oligonucleotide of claim 1 , wherein the one or more modifications comprises a substitution on the phosphate backbone with one or more nucleotide linkers to produce a modified oligonucleotide having one of the following nucleotide linking structures:

7 . The modified oligonucleotide of claim 1 , wherein the one or more modifications comprises a modified sugar structure and the oligonucleotide contains a modified nucleotide selected from the group consisting of:

8 . The modified oligonucleotide of claim 1 , wherein the one or more modifications comprises a moiety attached to the nucleobase and the moiety is selected from the group consisting of:

wherein:
n is a positive integer from 1 to 100.

9 . The modified oligonucleotide of claim 1 , wherein the modified nucleobase is selected from the group consisting of:

10 . The modified oligonucleotide of claim 1 , wherein the nucleobase comprises a nucleobase modification including one or more of: glutamic acid, polyglutamic acid, polylysine, polyamines, fluorine, G-quadraplexes, DNA origami, or macrocycles.

11 . The modified oligonucleotide of claim 1 , wherein the modified oligonucleotide additionally comprises a modified nucleotide containing a cyclic loop bridging the nucleobase and the phosphate backbone or bridging one portion of the phosphate backbone and an oxygen on the phosphate backbone.

12 . A modified nucleotide having one of the following structures:

wherein:
X is —O—, —CH 2 —, or —NH—;
Y is —O—, —S—, or —NH—;
Base is a nucleobase;
L 1 is a first linking group;
L 2 is a second linking group; and
Z is a cyclic loop modification containing at least one moiety to reduce, slow, or halt translocation of the nucleotide through a nanopore.

13 . The modified nucleotide of claim 12 , wherein Z includes a spacer and reporter, wherein the spacer and reporter each comprises one or more of the following moieties:
a) polypeptides having 10 to 100 repeating units b) pseudopeptides having 10 to 100 repeating units c) hydrophilic polymers having 10 to 100 repeating units d) hydrophobic polymers having 10 to 100 repeating units.

14 . The modified nucleotide of claim 12 , wherein Z includes one or more moieties to increase non-covalent interaction with the interior of the nanopore.

15 . The modified nucleotide of claim 14 , wherein the one or more moieties are selected from the group consisting of:

16 . The modified nucleotide of claim 12 , wherein Z includes one or more moieties to alter or reduce the net charge of the modified nucleotide.

17 . The modified nucleotide of claim 16 , the one or more moieties are selected from the group consisting of:

18 . The modified nucleotide of claim 12 , wherein Z includes one or more moieties to increase the bulk size of Z and increase steric hinderance.

19 . The modified nucleotide of claim 18 , wherein the one or more moieties are selected from the group consisting of:

20 . The modified nucleotide of claim 12 , wherein each of L 1 and L 2 independently comprises a conjugating moiety 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.

21 . A method for determining a sequence of a polynucleotide in a nanopore-based sequencing system, the method comprising:
providing a modified oligonucleotide comprising one or more modifications in at least one of a phosphate backbone, a nucleobases, or a sugar, wherein the one or more modifications impede translocation through a nanopore; applying a bias across a nanopore to cause the modified oligonucleotide to insert into and translocate through the nanopore; and (i) detecting and identifying a reporter moiety when the modified oligonucleotide passes through the nanopore; or (ii) detecting and identifying a base on the oligonucleotide when the oligonucleotide passes through the nanopore.

22 . The method of claim 21 , wherein the modified oligonucleotide comprises one or more modified nucleotides that comprise a cyclic loop, the cyclic loop having a first end attached to a first position of a nucleotide of the oligonucleotide and a second end attached to a second position of the nucleotide.

23 . The method of claim 22 , further comprising cleaving a cleavable bond on the one or more modified nucleotides between the first and the second positions, thereby elongating the one or more modified nucleotides to form an elongated polymer; and
i. detecting and identifying a

Assignees

• Illumina Inc

Inventors

• Mandell Jeffrey G
• Killian Jessica
• Yuen Tsz Ying
• Salam Abdul Sadeer Abd
• Neelakandan Ramesh
• Boyanov Boyan
• Yang Xiangyuan
• Moon John
• Teo Yin Nah

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6806

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

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07H19/10 Primary

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title