Method of nanopore sequencing of concatenated nucleic acids

Patent metadata

Field	Value
Publication number	US-12584170-B2
Application number	US-202117379931-A
Country	US
Kind code	B2
Filing date	Jul 19, 2021
Priority date	May 25, 2016
Publication date	Mar 24, 2026
Grant date	Mar 24, 2026

Abstract

The invention relates to a new method of characterising two or more target polynucleotides using a pore. The method involves sequentially attaching to a first polynucleotide one or more subsequent polynucleotides to form a concatenated polynucleotide.

First claim

The invention claimed is:

1 . A population of polynucleotide adaptors comprising a first polynucleotide adaptor and a second polynucleotide adaptor,
wherein the first polynucleotide adaptor and the second polynucleotide adaptor each comprise a first polynucleotide strand and a second polynucleotide strand, each strand having a 5′ end and a 3′ end, wherein: (a) a portion extending to the 3′ end of the first polynucleotide strand is complementary to a portion extending to the 5′ end of the second polynucleotide strand, and the complementary portions form a duplex; (b) a single stranded portion extending to the 5′ end of the first polynucleotide strand and a single stranded portion extending to the 3′ end of the second polynucleotide strand are not complementary and do not hybridise to one another; (c) the complementary portion extending to the 5′ end of the second polynucleotide strand of the first polynucleotide adaptor comprises a sequence that is capable, when the duplex is unwound, of hybridising to a sequence comprised in the single stranded portion extending to the 5′ end of the first polynucleotide strand of the second polynucleotide adaptor; and (d) the 5′ end of the first polynucleotide strand comprises a click reactive group and the 3′ end of the second polynucleotide strand comprises a complementary click reactive group.

2 . The population according to claim 1 , wherein a sequence comprised in the portion extending to the 5′ end of the second polynucleotide strand of the first polynucleotide adaptor and a sequence comprised in the single stranded portion extending to the 5′ end of the first polynucleotide strand of the second polynucleotide adaptor have a length of from 6 to 50 nucleotides.

3 . The population according to claim 1 , wherein one portion of a non-complementary region in the second polynucleotide strand of each polynucleotide adaptor forms a loop.

4 . The population according to claim 1 , wherein only one of the polynucleotide adaptors in the population comprises a molecular brake.

5 . The population according to claim 4 , wherein the molecular brake is or is derived from a polymerase, helicase, or exonuclease.

6 . The population according to claim 1 , wherein the duplex has a length of 6 to 200 base pairs.

7 . The population according to claim 1 , wherein the duplex comprises a blocker.

8 . The population according to claim 7 , wherein the blocker comprises iSp18.

9 . The population according to claim 1 , wherein the single stranded portion extending to the 5′ end of the first polynucleotide strand comprises a single stranded leader sequence.

10 . The population according to claim 9 , wherein the leader sequence is 10 to 150 nucleotides in length.

11 . The population according to claim 1 , wherein each polynucleotide adapter comprises one or more polynucleotide binding proteins.

12 . The population according to claim 11 , wherein the one or more polynucleotide binding proteins is attached to the first polynucleotide strand of each polynucleotide adapter.

13 . The population according to claim 1 , wherein each polynucleotide adaptor comprises one or more anchors.

14 . The population according to claim 13 , wherein the one or more anchors is cholesterol.

15 . A population of polynucleotide adaptors comprising a first polynucleotide adaptor and a second polynucleotide adaptor,
wherein the first polynucleotide adaptor and the second polynucleotide adaptor each comprise a first polynucleotide strand and a second polynucleotide strand, each strand having a 5′ end and a 3′ end, wherein: (a) a portion extending to the 3′ end of the first polynucleotide strand is complementary to a portion extending to the 5′ end of the second polynucleotide strand, and the complementary portions form a duplex; (b) a single stranded portion extending to the 5′ end of the first polynucleotide strand and a single stranded portion extending to the 3′ end of the second polynucleotide strand are not complementary and do not hybridise to one another; (c) the complementary portion extending to the 5′ end of the second polynucleotide strand of the first polynucleotide adaptor comprises a sequence that is capable, when the duplex is unwound, of hybridising to a sequence comprised in the single stranded portion extending to the 5′ end of the first polynucleotide strand of the second polynucleotide adaptor; and (d) the 5′ end of the first polynucleotide strand comprises a phosphatase.

16 . A kit for characterising two or more double stranded target polynucleotides, comprising the population of polynucleotide adaptors according to claim 1 and one or more of the following: a population of hairpin loops; a microparticle; one or more anchors capable of coupling a polynucleotide to a membrane; membrane components; and a magnet or electromagnet.

17 . The kit according to claim 16 , wherein the membrane components comprise components of an amphiphilic layer or a triblock copolymer membrane.

18 . The kit according to claim 16 , wherein the kit further comprises a transmembrane protein pore.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Heron Andrew John
• Garalde Daniel Ryan
• White James

Classifications

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

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