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

• US 10,443,097 B2
• Filed: 05/01/2015
• Issued: 10/15/2019
• Est. Priority Date: 05/02/2014
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• 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
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US15/308,252
• Publication Number:
  US 20170058338A1
• Time in Patent Office: 1,628 Days
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/157 Nanotubes or nanorods
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

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.

Claims

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 transmembrane pore which interacts with the polynucleotide binding protein and/or 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 transmembrane pore or 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 modification(s) alter the charge, sterics, hydrogen bonding, π

stacking or structure of the part of the transmembrane pore which interacts with the polynucleotide binding protein and/or the part of the polynucleotide binding protein which interacts with the transmembrane pore.

3. The method of claim 1, wherein the transmembrane pore is comprised of seven or more modified MspA monomers and the polynucleotide binding protein is Dda helicase or modified Dda helicase.

4. The method according to claim 3, wherein the part of the MspA monomer which interacts with the Dda helicase comprises modified amino acids at positions:

(a) 12, 14, 48, 52, 53, 54, 55, 56, 57, 58, 59, 60, 134, 135, 136, 137, 138, 139, 169 and 170 in SEQ ID NO;

2;

(b) 12, 14, 52, 54, 56, 57, 59, 134, 136, 138, 139 and 169 in SEQ ID NO;

2;

(c) 12, 14, 56, 57, 59, 134, 136, 139 and 169 in SEQ ID NO;

2;

(d) 56, 57, 59, 134, 136, 139 and 169 in SEQ ID NO;

2;

or

(e) 56, 57, 59, 134 and 139 in SEQ ID NO;

2.

5. The method according to claim 4, wherein the one or more of the seven or more monomers are modified such that they do not comprise aspartic acid (D) or glutamic acid (E) at one or more of positions 56, 57, 59, 134 and 139 of SEQ ID NO:

2.

6. The method according to claim 5, wherein the one or more of the seven or more monomers are modified such that they comprise one or more of

(a) D56N or D56R,

(b) E57N or E57R,

(c) E59N or E59R,

(d) D134N or D134R and

(e) E139N, E139R or E139K.

7. The method according to claim 4, wherein the MspA monomer further comprises amino acid substitutions selected from the group consisting of:

(a) D90N, D91N, D118R, D134R and E139K and optionally D93N;

(b) L88N, D90N, D91N, D93N, D118R, D134R, and E139K;

(c) G75S, G77S, L88N, D90N, D91N, D93N, D118R, Q126R, D134R, and E139K;

or

(d) G75S, G77S, L88N, D90N, D91N, D118R, Q126R, D134R, and E139K.

8. The method according to claim 4, wherein the MspA monomer further comprises amino acid deletions relative to SEQ ID NO:

2 selected from the group consisting of;

(a) 2, 4, 6, 8 or 10 of the amino acids at positions 72 to 82 of SEQ ID NO;

2 and

(b) 2, 4, 6, 8 or 10 of the amino acids at positions 111 to 121 of SEQ ID NO;

2.

9. The method according to claim 3, wherein the part of the Dda helicase which interacts with the MspA pore comprises modified amino acids at positions:

(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.

10. The method according to claim 9, wherein the modified Dda helicase further comprises an amino acid substitutions relative to SEQ ID NO:

24 at;

(a) E94C and A360C or

(b) E94C, A360C, C109A and C136A.