Modified helicases

Patent metadata

Field	Value
Publication number	US-11525126-B2
Application number	US-202017064329-A
Country	US
Kind code	B2
Filing date	Oct 6, 2020
Priority date	Jul 19, 2012
Publication date	Dec 13, 2022
Grant date	Dec 13, 2022

Abstract

The invention relates to modified helicases with reduced unbinding from polynucleotides. The helicases can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

First claim

The invention claimed is:

1. An NS3 helicase comprising a polynucleotide binding domain which comprises in at least one conformational state an opening through which a polynucleotide can unbind from the helicase, wherein the helicase is modified such that two or more parts of the helicase are connected using one or more linkers and wherein the helicase retains its ability to control the movement of the polynucleotide, wherein the linkers connect two amino acid residues that are located in different structural domains on the surface of the NS3 helicase surrounding the polynucleotide binding domain or wherein the linkers connect two amino acid residues on one or more loop regions connecting α-helices and β-strands of the NS3 helicase and wherein at least one amino acid of the two amino acid residues is substituted with cysteine, a non-natural amino acid or 4-azido-L-phenylalanine (Faz).

2. The NS3 helicase according to claim 1 , wherein the linkers connect two amino acid residues that are located in different structural domains on the surface of the NS3 helicase surrounding the polynucleotide binding domain.

3. The NS3 helicase according to claim 1 , wherein the linkers connect two amino acid residues on one or more loop regions connecting α-helices and β-strands of the NS3 helicase and/or are spatially located proximal to the polynucleotide binding domain.

4. The NS3 helicase according to claim 1 , wherein the one or more linkers are amino acid sequences and/or one or more chemical crosslinkers.

5. The NS3 helicase according to claim 1 , wherein the linkers comprise a polyethyleneglycol (PEG), polysaccharide, or polyamide.

6. The NS3 helicase according to claim 1 , wherein the linkers comprise a deoxyribonucleic acid (DNA) sequence, peptide nucleic acid (PNA), threose nucleic acid (TNA), or glycerol nucleic acid (GNA).

7. The NS3 helicase according to claim 1 , wherein one end or both ends of the one or more linkers are covalently attached to the helicase.

8. The NS3 helicase according to claim 1 , wherein the helicase has a covalently closed structure.

9. The NS3 helicase according to claim 1 , wherein the helicase further comprises a second set of two amino acid residues in different structural domains of the helicase surrounding the polynucleotide binding domain that are artificially covalently connected via a linkage between the two amino acid residues of the second set.

10. A complex comprising (i) the NS3 helicase according to claim 1 and (ii) a target polynucleotide bound to the polynucleotide binding domain, wherein two amino acid residues that are located in different structural domains on the surface of the NS3 helicase surrounding the polynucleotide binding domain are artificially covalently connected via a linkage between the two amino acid residues, such that the helicase has a covalently-closed structure.

11. The complex according to claim 10 , wherein the distance between the two amino acids is less than 50 Angstroms (Å), wherein the bound target polynucleotide is encircled by the covalently-closed structure.

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Heron Andrew
• Clarke Anthony
• Moysey Ruth
• Wallace Elizabeth Jayne
• Bruce Mark John
• Jayasinghe Lakmal
• Caprotti Domenico
• Soeroes Szabolcs
• Mcneill Luke
• Alves David Antoni
• Bowen Rebecca Victoria
• Milton John

Classifications

• C12Y599/01002

  DNA topoisomerase (5.99.1.2) · CPC title

• C12N9/90

  Isomerases (5.) · CPC title

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C12Y599/01003

  DNA topoisomerase (ATP-hydrolysing) (5.99.1.3) · CPC title

• C12Y306/04012

  DNA helicase (3.6.4.12) · CPC title