Alpha-hemolysin variants and uses thereof

Patent metadata

Field	Value
Publication number	US-10968480-B2
Application number	US-201916425743-A
Country	US
Kind code	B2
Filing date	May 29, 2019
Priority date	Apr 21, 2016
Publication date	Apr 6, 2021
Grant date	Apr 6, 2021

Abstract

Described herein are variants of alpha-hemolysin having at least one mutation, such as a mutation to a positive charge. In certain examples, the mutation is selected from V149K, E287R, H35G, T109K, P151K, K147N, E111N, M113A, or combinations thereof in the mature, wild-type alpha-hemolysin amino acid sequence. The α-hemolysin variants may also include a substitution at H144A and/or a series of glycine residues spanning residues 127 to 131 of the mature, wild-type alpha hemolysin. Also provided are nanopore assemblies including the alpha-hemolysin variants, the assembly having a decreased time-to-thread. The decreased time-to-thread, for example, increases DNA sequencing efficiency and accuracy.

First claim

What is claimed is:

1. An α-hemolysin variant having at least 90% sequence identity to one or more of SEQ ID NOs: 5-14, wherein said amino acid sequence has a set of substitutions relative to SEQ ID NO: 14 comprising at least one of the following substitution combinations:
(a) H35G+V149K;
(b) H35G+V149K+E287R;
(c) V149K+E287R;
(d) H35G+T109K;
(e) H35G+P151K;
(f) H35G+V149K+P151K;
(g) H35G+T109K+V149K;
(h) H35G+E111N+M113A+D127G+D128G+T129G+K131G+K147N+V149K;
(i) E111N+M113A+D127G+D128+T129G+K131G+K147N+V149K;
(j) H353G+T109K+V149K+P151K; and
(k) H35G+H144A+V149K+E287R.

2. The α-hemolysin variant of claim 1 , wherein said set of substitutions further comprise substitutions corresponding to positions of SEQ 10 NO: 14 selected from the group consisting of 1-6, 8-11, 13-22, 24-33, 35-37, 40, 44-51, 56, 62, 64-75, 79, 82, 83, 85, 87, 89-95, 97, 102-118, 120-132, 134-156, 158-164, 168, 170-191, 193, 194, 197-205, 207, 208, 210-216, 218, 221, 222, 224-229, 235-241, 244, 246, 250, 252, 253, 255, 257, 259-264, 266, 268-278, 280-283, 285-289, and 291-293.

3. The α-hemolysin variant of claim 1 , wherein said set of substitutions (a) through (j) further comprises an H144A substitution.

4. The α-hemolysin variant of claim 1 , wherein said set of substitutions further comprises an alanine, asparagine, or lysine substitution at one or more of residues 1-17 of SEQ ID NO: 14.

5. The α-hemolysin variant of claim 1 , wherein said set of substitutions (a) through (g), (j), and (k) further comprises a series of glycine residues at positions corresponding to each of residues 127-131 of SEQ ID NO: 14.

6. The α-hemolysin variant of claim 1 , wherein the amino acid sequence comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

7. The α-hemolysin variant of claim 1 , wherein the amino acid sequence consists of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

8. The α-hemolysin variant of claim 1 , further comprising a purification tag.

9. The α-hemolysin variant of claim 1 , further comprising a polymerase attached thereto.

10. The α-hemolysin variant of claim 9 , wherein the polymerase is a DNA polymerase.

11. The α-hemolysin variant of claim 10 , wherein the DNA polymerase is covalently attached to the isolated polypeptide.

12. A heptameric nanopore assembly comprising 7 alpha-hemolysin subunits arranged in a nanopore, wherein at least one of the seven alpha-hemolysin subunits comprises the isolated α-hemolysin variant of claim 1 and a DNA polymerase covalently attached to one of the seven alpha-hemolysin subunits.

13. The heptameric nanopore assembly of claim 12 , wherein the nanopore is a heteromeric pore.

14. The heptameric nanopore assembly of claim 12 , wherein the nanopore is a homomeric pore.

15. The heptameric nanopore assembly of claim 12 , wherein the nanopore has a decreased time to thread (TTT) relative to a heptameric nanopore assembly consisting of native alpha-hemolysins.

16. The heptameric nanopore assembly of claim 15 , wherein the nanopore has at least a 10% decreased TTT relative to the heptameric nanopore assembly consisting of native alpha-hemolysins.

17. The heptameric nanopore assembly of claim 15 , wherein the nanopore has at least a 50% decreased TTT relative to the heptameric nanopore assembly consisting of native alpha-hemolysins.

18. The heptameric nanopore assembly of claim 15 , wherein the nanopore has at least an 80% decreased TTT relative to the heptameric nanopore assembly consisting of native alpha-hemolysins.

19. A system for sequencing a nucleic acid comprising: a heptameric nanopore assembly according to claim 12 ; a chip comprising a plurality of sensing electrodes and a membrane that is disposed adjacent or in proximity to one or more of the sensing electrodes; and a set of negatively charged tagged nucleotides.

20. A method for sequencing a target nucleic acid sequence, comprising: providing a chip, the chip comprising a plurality of sensing electrodes and a membrane that is disposed adjacent or in proximity to the sensing electrodes; disposing, within the membrane, a heptameric nanopore assembly of claim 12 ; contacting the chip with a target nucleic acid sequence and a plurality of negatively charged tagged nucleotides; applying a voltage across the membrane; determining, by one or more of the sensing electrodes, one or more current changes associated with the heptameric nanopore assembly; and determining, with the aid of a computer processor and based on the one or more of the determined current changes associated with the heptameric nanopore assembly, a sequence for the target nucleic acid sequence.

21. The method of claim 20 , wherein the chip comprises a well and wherein the nanopore assembly is disposed within the membrane over the well.

22. The method of claim 20 , wherein the nucleic acid comprises a tag.

23. The method of claim 22 , wherein the tag is a negatively charged tag.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Craig Timothy K
• Ambroso Mark R
• Harris Corissa J
• Dipietro Matthew M
• Zou Yaozhong
• Porter Marshall W

Classifications

• C12Q1/689

  for bacteria · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N33/48721

  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