Modified helicases

Patent metadata

Field	Value
Publication number	US-12258591-B2
Application number	US-202218047019-A
Country	US
Kind code	B2
Filing date	Oct 17, 2022
Priority date	Oct 18, 2013
Publication date	Mar 25, 2025
Grant date	Mar 25, 2025

Abstract

The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Dda helicase. The helicase controls the movement of the target polynucleotide through the pore. The invention also relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

First claim

The invention claimed is:

1. A kit for characterising a target polynucleotide comprising:
(a) a pore and
(b) a helicase comprising:
(i) a first polypeptide comprising the pin domain and the 1A domain from a Dda helicase and not comprising any other domains from a Dda helicase, wherein at least one cysteine residue and/or at least one non-natural amino acid have been introduced into the pin domain and/or the 1A domain, and
(ii) a second polypeptide comprising the 2A domain, the tower domain and the hook domain from a Dda helicase and not comprising any other domains from a Dda helicase, wherein at least one cysteine residue and/or at least one non-natural amino acid have been introduced into the tower domain, wherein the helicase has the ability to control the movement of a polynucleotide;

wherein two or more of the introduced cysteines and/or non-natural amino acids are connected to one another;
wherein said first polypeptide is covalently attached to said second polypeptide; and
wherein the pore is (1) a transmembrane protein pore, (2) a solid state pore, or (3) an oligomer pore.

2. The kit of claim 1 , wherein the at least one non-natural amino acid in the first polypeptide and/or the at least one non-natural amino acid in the second polypeptide is 4-azido-L-phenylalanine (Faz).

3. The kit of claim 1 , wherein the helicase comprises at least one introduced cysteine residue and at least one introduced non-natural amino acid.

4. The kit of claim 3 , wherein the at least one introduced cysteine residue is connected to the at least one introduced non-natural amino acid.

5. The kit of claim 1 , wherein the first polypeptide is covalently attached to the second polypeptide via an amino acid.

6. The kit of claim 1 , wherein the helicase is further modified to increase or decrease its surface charge.

7. The kit of claim 1 , wherein the helicase is further modified by the removal of one or more native cysteine residues.

8. The kit of claim 7 , wherein the one or more cysteine residues are removed by substitution.

9. The kit of claim 1 , wherein the helicase is covalently attached to the pore.

10. The kit of claim 1 , wherein the pore is derived from Msp.

11. The kit of claim 1 , wherein the pore is a MspA pore.

12. The kit of claim 1 , further comprising an additional polynucleotide binding moiety, wherein the helicase is attached to the polynucleotide binding moiety.

13. The kit of claim 1 , wherein the kit comprises two or more of the helicases.

14. The kit of claim 1 , further comprising an additional polynucleotide binding moiety, wherein the pore is attached to the polynucleotide binding moiety.

15. The kit of claim 1 , wherein the kit further comprises a chip comprising a amphiphilic membrane.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Bruce Mark
• Heron Andrew John
• Moysey Ruth
• Soeroes Szabolcs
• Wallace Elizabeth Jayne
• White James

Classifications

• C12Y306/04012

  DNA helicase (3.6.4.12) · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12N9/14 Primary

  Hydrolases (3) · CPC title