Method of characterising a target polypeptide using a nanopore

Patent metadata

Field	Value
Publication number	US-2023024319-A1
Application number	US-202017781469-A
Country	US
Kind code	A1
Filing date	Dec 1, 2020
Priority date	Dec 2, 2019
Publication date	Jan 26, 2023
Grant date	—

Abstract

Provided herein are methods of characterising a target polypeptide as it moves with respect to a nanopore. Also provided are related kits, systems and apparatuses for carrying out such methods.

First claim

1 . A method of characterising a target polypeptide, comprising
conjugating the target polypeptide to a polynucleotide to form a polynucleotide-polypeptide conjugate; contacting the conjugate with a polynucleotide-handling protein capable of controlling the movement of the polynucleotide with respect to a nanopore; and taking one or more measurements characteristic of the polypeptide as the conjugate moves with respect to the nanopore,
thereby characterising the polypeptide.

2 . A method according to claim 1 , wherein the nanopore is modified to extend the distance between the polynucleotide-handling protein and a constriction region of the nanopore.

3 . A method according to claim 1 or claim 2 , comprising separating the polypeptide-handling protein from the nanopore using a displacer unit thereby extending the distance between the polynucleotide-handling protein and the nanopore.

4 . A method according to claim 3 , wherein the displacer unit comprises one or more proteins.

5 . A method according to any one of the preceding claims, wherein the polynucleotide-handling protein is modified to extend the distance from the active site of the polynucleotide-handling protein to the nanopore.

6 . A method according to any one of the preceding claims, wherein the polynucleotide-handling protein is capable of remaining bound to the conjugate when the portion of the conjugate in contact with the active site of the polynucleotide-handling protein comprises a polypeptide.

7 . A method according to any one of the preceding claims, wherein the polynucleotide-handling protein is modified to prevent it from disengaging from the conjugate when the polynucleotide-handling protein contacts a portion of the conjugate comprising a polypeptide.

8 . A method according to any one of the preceding claims, wherein the polynucleotide-handling protein is modified to wholly or partially close an opening existing in at least one conformation state of the unmodified protein through which a polynucleotide strand can unbind.

9 . A method according to any one of the preceding claims, wherein the polynucleotide-handling protein is a helicase.

10 . A method according to any one of the preceding claims, wherein the conjugate comprises a plurality of polypeptide sections and/or a plurality of polynucleotide sections.

11 . A method according to any one of the preceding claims, wherein the polypeptide has a length of from 2 to about 50 peptide units.

12 . A method according to any one of the preceding claims, wherein the polypeptide is held in a linearized form.

13 . A method according to any one of the preceding claims, wherein the polynucleotide has a length of from about 10 to about 1000 nucleotides.

14 . A method according to any one of the preceding claims, wherein one or more adapters and/or one or more tethers and/or one or more anchors are attached to the polynucleotide in the conjugate.

15 . A method according to any one of the preceding claims, wherein:
i) the polynucleotide-handling protein is located on the cis side of the nanopore and the polynucleotide-handling protein controls the movement of the conjugate from the cis side of the nanopore to the trans side of the nanopore; or ii) the polynucleotide-handling protein is located on the trans side of the nanopore and the polynucleotide-handling protein controls the movement of the conjugate from the trans side of the nanopore to the cis side of the nanopore.

16 . A method according to claim 15 , wherein the polynucleotide-handling protein is located on the cis side of the nanopore and the polynucleotide-handling protein controls the movement of the polynucleotide from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the polypeptide through the nanopore.

17 . A method according to claim 15 , wherein the polynucleotide-handling protein is located on the trans side of the nanopore and the polynucleotide-handling protein controls the movement of the polynucleotide from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the polypeptide through the nanopore.

18 . A method according to any one of claims 1 to 15 , wherein the conjugate comprises one or more structures of the form L-{P-N}-P m , wherein:
L is a leader, wherein L is optionally an N moiety;
P is a polypeptide;
N comprises a polynucleotide; and
m is 0 or 1;

and wherein the method comprises threading the leader (L) through the nanopore thereby contacting the polypeptide (P) with the nanopore; and
i) the polynucleotide-handling protein is located on the cis side of the nanopore and the method comprises allowing the polynucleotide-handling protein to control the movement of the polynucleotide moiety (N) from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of the polypeptide (P) through the nanopore; or
ii) the polynucleotide-handling protein is located on the trans side of the nanopore and the method comprises allowing the polynucleotide-handling protein to control the movement of the polynucleotide moiety (N) from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the polypeptide (P) through the nanopore.

19 . A method according to claim 18 , wherein the conjugate comprises one or more structures of the form L-P 1 -N-{P-N}n-P m , wherein:
n is a positive integer; L is a leader, wherein L is optionally an N moiety; each P, which may be the same or different, is a polypeptide; each N, which may be the same or different, comprises a polynucleotide; and m is 0 or 1;
and wherein the method comprises threading the leader (L) through the nanopore thereby contacting polypeptide (P 1 ) with the nanopore, and
i) the polynucleotide-handling protein is located on the cis side of the nanopore and the method comprises allowing the polynucleotide-handling protein to control the movement of each polynucleotide (N) sequentially from the cis side of the nanopore to the trans side of the nanopore, thereby controlling the movement of each polypeptide (P) sequentially through the nanopore; or
ii) the polynucleotide-handling protein is located on the trans side of the nanopore and the method comprises allowing the polynucleotide-handling protein to control the movement of each polynucleotide (N) sequentially from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of each polypeptide (P) sequentially through the nanopore

20 . A method according to any one of claims 1 to 14 , wherein:
i) the polynucleotide-handling protein is located on the cis side of the nanopore and the polynucleotide-handling protein controls the movement of the conjugate from the trans side of the nanopore to the cis side of the nanopore; or
ii) the polynucleotide-handling protein is located on the trans side of the nanopore and the polynucleotide-handling protein controls the movement of the conjugate from the cis side of the nanopore to the trans side of the nanopore.

21 . A method according to claim 20 , wherein the polynucleotide-handling protein is located on the cis side of the nanopore and the polynucleotide-handling protein controls the movement of the polynucleotide from the trans side of the nanopore to the cis side of the nanopore, thereby controlling the movement of the polypeptide through the nanopore.

22 . A method according to claim

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Heron Andrew John
• Graham James Edward
• Strycharska Melania Slawa

Classifications

• G01N33/6818 Primary

  Sequencing of polypeptides · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• G01N2458/00

  Labels used in chemical analysis of biological material · CPC title

• C12N2310/3517

  Marker; Tag · CPC title

• G01N33/48721 Primary

  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