Method of improving the movement of a target polynucleotide with respect to a transmembrane pore

Patent metadata

Field	Value
Publication number	US-10844432-B2
Application number	US-201916572869-A
Country	US
Kind code	B2
Filing date	Sep 17, 2019
Priority date	May 2, 2014
Publication date	Nov 24, 2020
Grant date	Nov 24, 2020

Abstract

The invention relates to improving the movement of a target polynucleotide with respect to a transmembrane pore when the movement is controlled by a polynucleotide binding protein. The invention also relates to improved transmembrane pores and polynucleotide binding proteins.

First claim

The invention claimed is:

1. A method of characterizing a target polynucleotide, comprising:
(a) providing a transmembrane pore and a polynucleotide binding protein in which a part of the polynucleotide binding protein which interacts with the transmembrane pore has been modified, wherein the modification comprises an amino acid substitution, insertion, or deletion relative to an unmodified polynucleotide binding protein;
(b) contacting the transmembrane pore and polynucleotide binding protein provided in (a) with the target polynucleotide such that the polynucleotide binding protein controls the movement of the polynucleotide with respect to the transmembrane pore; and
(c) taking one or more electrical or optical measurements as the polynucleotide moves with respect to the transmembrane pore.

2. The method according to claim 1 , wherein the surface of the polynucleotide binding protein which interacts with the transmembrane pore has been modified.

3. The method according to claim 1 , wherein the modification(s) alter the charge, sterics, hydrogen bonding, 7C stacking, or structure of the part of the polynucleotide binding protein which interacts with the transmembrane pore.

4. The method according to claim 1 , wherein the polynucleotide binding protein is a helicase.

5. The method according to claim 1 , wherein the polynucleotide binding protein is a He1308 helicase, a RecD helicase, a TraI helicase, a TrwC helicase, a XPD helicase, or a Dda helicase.

6. The method according to claim 1 , wherein the polynucleotide binding protein is a Dda helicase.

7. The method according to claim 1 , wherein the polynucleotide binding protein comprises the sequence shown in SEQ ID NO: 24.

8. The method according to claim 7 , wherein the part of the polynucleotide binding protein which interacts with the transmembrane pore comprises the amino acids at
(a) positions 1, 2, 3, 4, 5, 6, 51, 176, 177, 178, 179, 180, 181, 185, 189, 191, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 207, 208, 209, 210, 211, 212, 213, 216, 219, 220, 221, 223, 224, 226, 227, 228, 229, 247, 254, 255, 256, 257, 258, 259, 260, 261, 298, 300, 304, 308, 318, 319, 321, 337, 347, 350, 351, 405, 415, 422, 434, 437, and 438 in SEQ ID NO: 24;
(b) positions 1, 2, 4, 51, 177, 178, 179, 180, 185, 193, 195, 197, 198, 199, 200, 202, 203, 204, 207, 208, 209, 210, 211, 212, 216, 221, 223, 224, 226, 227, 228, 229, 254, 255, 256, 257, 258, 260, 304, 318, 321, 347, 350, 351, 405, 415, 422, 434, 437 and 438 in SEQ ID NO: 24; or
(c) positions 1, 2, 178, 179, 180, 185, 195, 197, 198, 199, 200, 202, 203, 207, 209, 210, 212, 216, 221, 223, 226, 227, 255, 258, 260, 304, 350 and 438 in SEQ ID NO: 24.

9. The method according to claim 7 , wherein the polynucleotide binding protein comprises one of the following sets of amino acid substitutions relative to SEQ ID NO: 24:
(a) E94C and A360C; or
(b) E94C, A360C, C109A and C136A.

10. The method according to claim 1 , further wherein a part of the transmembrane pore which interacts with the polynucleotide binding protein has been modified, wherein the modification comprises an amino acid substitution, insertion, or deletion relative to an unmodified transmembrane pore.

11. A method of characterizing a target polynucleotide, comprising:
(a) providing a transmembrane pore and a Dda helicase in which a part of the Dda helicase which interacts with the transmembrane pore has been modified, wherein the modification comprises an amino acid substitution, insertion, or deletion relative to an unmodified Dda helicase;
(b) contacting the transmembrane pore and the Dda helicase provided in (a) with the target polynucleotide such that the Dda helicase controls the movement of the polynucleotide with respect to the transmembrane pore; and
(c) taking one or more electrical or optical measurements as the polynucleotide moves with respect to the transmembrane pore.

12. The method according to claim 11 , wherein the Dda helicase comprises the sequence shown in SEQ ID NO: 24.

13. The method according to claim 12 , wherein the part of the Dda helicase which interacts with the transmembrane pore comprises the amino acids at
(a) positions 1, 2, 3, 4, 5, 6, 51, 176, 177, 178, 179, 180, 181, 185, 189, 191, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 207, 208, 209, 210, 211, 212, 213, 216, 219, 220, 221, 223, 224, 226, 227, 228, 229, 247, 254, 255, 256, 257, 258, 259, 260, 261, 298, 300, 304, 308, 318, 319, 321, 337, 347, 350, 351, 405, 415, 422, 434, 437 and 438 in SEQ ID NO: 24;
(b) positions 1, 2, 4, 51, 177, 178, 179, 180, 185, 193, 195, 197, 198, 199, 200, 202, 203, 204, 207, 208, 209, 210, 211, 212, 216, 221, 223, 224, 226, 227, 228, 229, 254, 255, 256, 257, 258, 260, 304, 318, 321, 347, 350, 351, 405, 415, 422, 434, 437 and 438 in SEQ ID NO: 24; or
(c) positions 1, 2, 178, 179, 180, 185, 195, 197, 198, 199, 200, 202, 203, 207, 209, 210, 212, 216, 221, 223, 226, 227, 255, 258, 260, 304, 350 and 438 in SEQ ID NO: 24.

14. The method according to claim 12 , wherein the polynucleotide binding protein comprises one of the following sets of amino acid substitutions relative to SEQ ID NO: 24:
(a) E94C and A360C; or
(b) E94C, A360C, C109A and C136A.

15. The method according to claim 11 , further wherein a part of the transmembrane pore which interacts with the Dda helicase has been modified, wherein the modification comprises an amino acid substitution, insertion, or deletion relative to an unmodified transmembrane pore.

16. A method of characterizing a target polynucleotide, comprising:
(a) providing a transmembrane pore and a polynucleotide binding protein in which a part of the polynucleotide binding protein which interacts with the transmembrane pore has been modified and a part of the transmembrane pore which interacts with the polynucleotide binding protein has been modified,
wherein the modification of the polynucleotide binding protein comprises an amino acid substitution, insertion, or deletion relative to an unmodified polynucleotide binding protein, and
wherein the modification of the transmembrane pore comprises an amino acid substitution, insertion, or deletion relative to an unmodified transmembrane pore;
(b) contacting the transmembrane pore and polynucleotide binding protein provided in (a) with the target polynucleotide such that the polynucleotide binding protein controls the movement of the polynucleotide with respect to the transmembrane pore; and
(c) taking one or more electrical or optical measurements as the polynucleotide moves with respect to the transmembrane pore.

17. The method according to claim 16 , wherein the polynucleotide binding protein is a helicase.

18. The method according to claim 16 , wherein the polynucleotide binding protein is a He1308 helicase, a RecD helicase, a TraI helicase, a TrwC helicase, a XPD helicase, or a Dda helicase.

19. The method according to claim 16 , wherein the polynucleotide binding protein comprises the sequence shown in SEQ ID NO: 24.

20. The method according to claim 19 , wherein the polynucleotide binding protein comprises one of the following sets of amino acid substitutions relative to SEQ ID NO: 24:
(a) E94C and A360C; or
(b) E94C, A360C, C109A and C136A.

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Jayasinghe Lakmal
• Wallace Elizabeth Jayne
• Pugh Jonathan Bankes
• Hambley Richard George
• Wood Neil Roger
• Brown Clive Gavin
• White James
• Heron Andrew John
• Bruce Mark John
• Youd Christopher Peter
• Bowen Rebecca Victoria

Classifications

• C07K14/35 Primary

  from Mycobacteriaceae (F) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title