IP Library › Granted Patent US 9,170,230
Granted Patent B2
US 9,170,230 · App. 14/215,871 · Granted Oct 27, 2015

MSP nanopores and related methods

Inventors: Jens H. Gundlach (Seattle, WA); Michael Niederweis (Homewood, AL); Thomas Z. Butler (Seattle, WA); Mikhail Pavlenok (Birmingham, AL); Mark A. Troll (Seattle, WA); Suja Sukumaran (Dublin, CA)
Assignees: University of Washington; The UAB Research Foundation
G01N27/44704C07K14/35C12N13/00C12Q1/6869G01N33/48721G01N33/6872A61K39/04
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Quick Facts
Patent No.
US 9,170,230
App. No.
14/215,871
Granted
Oct 27, 2015
Kind
B2
Abstract

Provided herein are Mycobacterium smegmatis porin nanopores, systems that comprise these nanopores, and methods of using and making these nanopores. Such nanopores may be wild-type MspA porins, mutant MspA porins, wild-type MspA paralog porins, wild-type MspA homolog porins, mutant MspA paralog porins, mutant MspA homolog porins, or single-chain Msp porins. Also provided are bacterial strains capable of inducible Msp porin expression.

Claims (31)

1. A mutant Mycobacterium smegmatis porin A (MspA) monomer comprising a variant of the sequence in SEQ ID NO: 1, wherein the variant has at least 95% sequence identity to SEQ ID NO: 1, and wherein the variant comprises (i) mutations such that amino acid positions 90, 91, and 93 contain amino acids with a neutral charge and (ii) one or more mutations at the following amino acid positions: 88, 105, 108, 118, 126, 134, 138 or 139.

2. The mutant MspA monomer of claim 1 , wherein the variant comprises mutations at amino acid positions 118, 134, and 139.

3. The mutant MspA monomer of claim 1 , wherein the variant comprises the following mutations: D90N, D91N, and D93N.

4. The mutant MspA monomer of claim 1 , wherein the variant comprises the following mutations: D90N, D91N, D93N, D118R, D134R, and E139K.

5. The mutant MspA monomer of claim 1 , wherein the variant comprises the following mutations: D90N/Q/Y, D91N/Q/Y, and D93N/Q.

6. The mutant MspA monomer of claim 1 , wherein the variant comprises the mutation D93N.

7. The mutant MspA monomer of claim 1 , wherein the variant comprises one or more of the following mutations: L88W, 1105W, N108W, D118R, Q126R, D134R, A138P, and E139A/K.

8. The mutant MspA monomer of claim 1 , wherein the variant comprises a mutation at amino acid position 88.

9. The mutant MspA monomer of claim 1 , wherein the variant comprises a mutation at amino acid position 126.

10. A mutant Mycobacterium smegmatis porin A (MspA) monomer comprising a variant of the sequence in SEQ ID NO: 1, wherein the variant has at least 95% sequence identity to SEQ ID NO: 1, and wherein the variant comprises the following mutations: D90N/Q/Y, D91N/Q/Y, and D93N/Q.

11. The mutant MspA monomer of claim 10 , wherein the variant further comprises one or more of the following mutations: L88W, 1105W, N108W, D118R, D134R, A138P, and E139A/K.

12. The mutant MspA monomer of claim 10 , wherein the variant further comprises one or more mutations at the following positions: 88, 105, 108, 118, 126, 134, and 139.

13. The mutant MspA monomer of claim 10 , wherein the variant further comprises a mutation at amino acid position 88.

14. The mutant MspA monomer of claim 10 , wherein the variant further comprises a mutation at amino acid position 126.

15. A mutant Mycobacterium smegmatis porin A (MspA) monomer for nucleic acid sequencing, the mutant MspA comprising a variant of the sequence in SEQ ID NO: 1, wherein the variant consists essentially of the following mutations: D90N/Q/Y, D91N/Q/Y, D93N/Q, optionally D118R, optionally D134R, and optionally E139R/K.

16. The mutant MspA monomer of claim 15 , wherein the mutation D90N/Q/Y is D90N, wherein the mutation D91N/Q/Y is D91N, and wherein the mutation D93N/Q is D93N.

17. The mutant MspA monomer of claim 15 , wherein the variant consists essentially of the following mutations: D90N, D91N, D93N, D118R, D134R, and E139K.

18. A mutant Mycobacterium smegmatis porin A (MspA) monomer comprising a variant of the sequence in SEQ ID NO: 1, and wherein the variant comprises (i) mutations such that amino acid positions 90, 91, and 93 contain amino acids with neutral charge and (ii) one or more mutations such that at least one of amino acid positions 118, 134, and 139 contain an amino acid with positive charge.

19. The mutant MspA monomer of claim 18 , wherein the variant comprises a mutation such that amino acid position 118 contains an amino acid with positive charge.

20. The mutant MspA monomer of claim 18 , wherein the variant comprises mutations such that amino acid positions 118, 134, and 139 contain amino acids with positive charge.

21. The mutant MspA monomer of claim 18 , wherein the variant further comprises a mutation at position 88.

22. The mutant MspA monomer of claim 18 , wherein the variant comprises a chemically-modified amino acid.

23. The mutant MspA monomer of claim 18 , wherein the variant comprises the mutation D118R.

24. The mutant MspA monomer of claim 18 , wherein the variant comprises the following mutations: D118R and E139K.

25. The mutant MspA monomer of claim 18 , wherein the variant comprises the following mutations: D118R, D134R, and E139K.

26. The mutant MspA monomer of claim 25 , wherein the variant further comprises a mutation at position 88.

27. The mutant MspA monomer of claim 18 , wherein the variant comprises the following mutations: D90N, D91N, D93N, and D118R.

28. The mutant MspA monomer of claim 18 , wherein the variant comprises the following mutations: D90N, D91N, D93N, D118R, and E139K.

29. The mutant MspA monomer of claim 18 , wherein the variant comprises the following mutations: D90N, D91N, D93N, D118R, D134R, and E139K.

30. The mutant MspA monomer of claim 29 , wherein the variant further comprises a mutation at position 88.

31. A mutant Mycobacterium smegmatis porin A (MspA) monomer comprising a variant of the sequence in SEQ ID NO: 1, and wherein the variant comprises the following mutations: D90N, D91N, D93N, and Q126R.

Continuity (4)
Continuation 13069187 · Mar 22, 2011
Continuation PCTUS2009057915 · Sep 22, 2009
Provisional Application 61098938 · Sep 22, 2008
Related Publication 20140309402A1 · Oct 16, 2014