Alpha-hemolysin variants and uses thereof

Patent metadata

Field	Value
Publication number	US-10351908-B2
Application number	US-201715492214-A
Country	US
Kind code	B2
Filing date	Apr 20, 2017
Priority date	Apr 21, 2016
Publication date	Jul 16, 2019
Grant date	Jul 16, 2019

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 (α-HL) variant having at least 80% sequence identity to SEQ ID NO: 14 and comprising an amino acid substitution corresponding to any one of T109K, V149K, P151K, E287R, or combinations thereof in SEQ ID NO: 14.

2. The α-hemolysin variant according to claim 1 , wherein the variant has a sequence having at least 90%, 95%, 98%, or more sequence identity to the sequence set forth as SEQ ID NO: 14.

3. The α-hemolysin variant according to claim 1 , wherein the variant further comprises a substitution at H144A of SEQ ID NO: 14.

4. The α-hemolysin variant according to claim 1 , wherein the variant further comprises two or more glycine residues at residues 127 through 131.

5. The α-hemolysin variant according to claim 1 , wherein the substitution is V149K.

6. The α-hemolysin variant according to claim 1 , wherein the substitution is E287R.

7. The α-hemolysin variant to claim 1 , wherein the substitution is T109K.

8. The α-hemolysin variant to claim 1 , wherein the substitution is P151K.

9. The α-hemolysin variant to claim 1 , wherein the variant further comprises one or more substitutions substitution is selected from the group consisting of H35G, E111N, M113A, H144A and K147N of SEQ ID NO: 14.

10. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G, H144A, and V149K (SEQ ID NO: 4).

11. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G, H144A, V149K, and E287R (SEQ ID NO: 5).

12. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions V149K and E287R (SEQ ID NO: 6).

13. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G and T109K (SEQ ID NO: 7).

14. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G and P151K (SEQ ID NO: 8).

15. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G, V149K, and P151K (SEQ ID NO: 9).

16. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G, T109K, and V149K (SEQ ID NO: 10).

17. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G, E111N, M113A, K147N, and V149K and further comprising glycine at each of residues 127 through 131 (SEQ ID NO: 11).

18. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions E111N, M113A, K147N, and V149K and further comprising glycine at each of residues 127 through 131 (SEQ ID NO: 12).

19. The α-hemolysin variant according to claim 9 , wherein the variant comprises amino acid substitutions H35G, T109K, V149K, and P151K (SEQ ID NO: 10).

20. The α-hemolysin variant according to any of claims 1 , 3 - 6 , 7 - 9 , 2 , and 10 - 19 , wherein the α-hemolysin variant is covalently bound to a DNA polymerase.

21. The α-hemolysin variant according to claim 20 , wherein said variant is bound to the DNA polymerase via an isopeptide bond.

22. A heptameric nanopore assembly comprising at least one α-hemolysin variant according to any one of claims 1 , 3 - 6 , 7 - 9 , 2 , and 10 - 19 .

23. The heptameric nanopore assembly according to claim 22 , wherein the time to thread (TTT) is decreased.

24. The heptameric nanopore assembly according to claim 23 , wherein the TTT is decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more as compared to a heptameric nanopore assembly consisting of native alpha-hemolysin.

25. A nucleic acid encoding the α-hemolysin variant according to any one of claims 1 , 3 - 6 , 7 - 9 , 2 , and 10 - 19 .

26. The nucleic acid molecule of claim 25 , wherein said nucleic acid molecule is derived from Staphylococcus aureus (SEQ ID NO: 1).

27. A vector comprising a nucleic acid encoding an alpha-hemolysin variant according to claim 25 .

28. A host cell transformed with the vector of claim 27 .

29. A method of producing an α-hemolysin variant comprising the steps of:
(a) culturing the host cell according to claim 28 in a suitable culture medium under suitable conditions to produce alpha-hemolysin variant and
(b) obtaining said produced alpha-hemolysin variant.

30. A method for detecting a target molecule, comprising:
(a) providing a chip comprising a heptameric nanopore assembly according to claim 22 in a membrane that is disposed adjacent or in proximity to a sensing electrode;
(b) directing a nucleic acid molecule through said nanopore, wherein said nucleic acid molecule is associated with a reporter molecule, wherein said nucleic acid molecule comprises an address region and a probe region, wherein said reporter molecule is associated with said nucleic acid molecule at said probe region, and wherein said reporter molecule is coupled to a target molecule;
(c) sequencing said address region while said nucleic acid molecule is directed through said nanopore to determine a nucleic acid sequence of said address region; and
(d) identifying, with the aid of a computer processor, said target molecule based upon a nucleic acid sequence of said address region determined in (c).

Assignees

• Roche Sequencing Solutions Inc

Inventors

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

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C07K14/31

  from Staphylococcus (G) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N2333/31

  from Staphylococcus (G) · 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