Msp nanopores and related methods

Patent metadata

Field	Value
Publication number	US-9624275-B2
Application number	US-201414318072-A
Country	US
Kind code	B2
Filing date	Jun 27, 2014
Priority date	Sep 22, 2008
Publication date	Apr 18, 2017
Grant date	Apr 18, 2017

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.

First claim

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for determining an identity of one or more nucleotides in a nucleic acid, the method comprising:
translocating the nucleic acid from a first conductive liquid medium to a second conductive liquid medium in liquid communication through a mutant Mycobacterium smegmatis porin A (MspA) comprising a mutant MspA monomer with an amino acid sequence of SEQ ID NO:1 and further comprising (i) mutations such that positions 90 and 91 contain amino acids with a neutral charge, and (ii) one or more mutations adding a positively charged amino acid to a vestibule or entrance of the MspA;
measuring an ion current between the first conductive liquid medium and the second conductive liquid medium as nucleotides in the nucleic acid pass through the mutant MspA, thereby producing current patterns in the measured ion current; and
determining the identity of one or more nucleotides in the nucleic acid based on reference current patterns from known nucleotides.

2. The method of claim 1 , wherein the mutant MspA further comprises a second mutant MspA monomer, wherein the mutant MspA monomer has a different amino acid sequence than the second mutant MspA monomer.

3. The method of claim 1 , wherein the mutant MspA further comprises a second mutant MspA monomer, wherein the mutant MspA monomer is connected to a second mutant MspA monomer by one or more amino acid linkers.

4. The method of claim 1 , wherein translocating the nucleic acid from the first conductive liquid medium to the second conductive liquid medium in liquid communication through the mutant MspA comprises using a molecular motor to regulate a rate of translocation for the nucleic acid translocating through the mutant MspA.

5. The method of claim 4 , wherein the molecular motor is a helicase.

6. The method of claim 4 , wherein the molecular motor is a polymerase.

7. The method of claim 4 , wherein the nucleic acid has an average translocation velocity through the mutant MspA of less than 1 nucleotide/μs.

8. The method of claim 1 , wherein the mutant MspA monomer comprises a chemically modified amino acid.

9. The method of claim 1 , wherein the mutant MspA comprises a mutant MspA monomer, wherein the mutant MspA monomer does not include mutations in the periplasmic loop.

10. The method of claim 1 , wherein the nucleic acid comprises a hairpin, and wherein translocating the nucleic acid from the first conductive liquid medium to the second conductive liquid medium in liquid communication through the mutant MspA comprises denaturing the hairpin of the nucleic acid.

11. A method for determining an identity of one or more nucleotides in a nucleic acid, the method comprising:
introducing the nucleic acid from a first conductive liquid medium into a tunnel of a mutant Mycobacterium smegmatis porin A (MspA), wherein the mutant MspA is positioned between a first conductive liquid medium and a second conductive liquid medium, and wherein the mutant MspA comprises a mutant MspA monomer with an amino acid sequence of SEQ ID NO:1 and further comprising (i) mutations such that positions 90 and 91 contain amino acids with a neutral charge, and (ii) one or more mutations adding a positively charged amino acid to a vestibule or entrance of the MspA;
measuring an ion current between the first conductive liquid medium and the second conductive liquid medium as nucleotides in the nucleic acid occupy the tunnel of the mutant MspA, thereby producing current patterns in the measured ion current; and
determining the identity of one or more nucleotides in the nucleic acid occupying the tunnel of the mutant MspA based on reference current patterns from known nucleotides.

12. The method of claim 11 , wherein the mutant MspA monomer comprises the following mutations of SEQ ID NO:1: D90N, D91N, D93N, D118R, D134R, and E139K.

13. The method of claim 1 , wherein the mutant MspA monomer comprises the following mutations of SEQ ID NO:1: D90N/Q/Y and D91N/Q/Y.

14. The method of claim 1 , wherein the mutant MspA monomer comprises one or more of the following mutations of SEQ ID NO:1: D90N and D91N.

15. The method of claim 1 , wherein the mutant MspA monomer comprises one or more of the following mutations of SEQ ID NO:1: D90T and D91T.

16. The method of claim 1 , wherein the mutant MspA monomer comprises the following mutations of SEQ ID NO:1: D90N and D91N.

17. The method of claim 1 , wherein the mutant MspA monomer comprises the following mutations of SEQ ID NO:1: D90Q and D91Q.

18. The method of claim 1 , wherein the mutant MspA monomer does not have a mutation at position 93 of SEQ ID NO:1.

19. The method of claim 1 , wherein the mutant MspA monomer further comprises a mutation such that position 93 of SEQ ID NO:1 contains an amino acid with a neutral charge.

20. The method of claim 19 , wherein the mutant MspA monomer comprises the following mutation of SEQ ID NO:1: D93N/Q.

21. The method of claim 19 , wherein the mutant MspA monomer comprises one or more of the following mutations of SEQ ID NO:1: D90N/Q/Y, D91N/Q/Y, and D93N/Q.

22. The method of claim 19 , wherein the mutant MspA monomer comprises the following mutations of SEQ ID NO:1: D90N/Q/Y, D91N/Q/Y, and D93N/Q.

23. The method of claim 19 , wherein the mutant MspA monomer comprises the following mutations of SEQ ID NO:1: D90N, D91N, and D93N.

24. The method of claim 19 , wherein the mutant MspA monomer comprises the following mutations of SEQ ID NO:1: D90Q, D91Q, and D93N.

25. The method of claim 1 , wherein the mutant MspA monomer comprises at least one mutation such that a negatively charged amino acid is replaced by a positively charged amino acid in the vestibule of the mutant MspA.

26. The method of claim 1 , wherein the mutant MspA monomer comprises one or more mutations at the following amino acid positions of SEQ ID NO:1: 88, 105, 108, 118, 134, and 139.

27. The method of claim 1 , wherein the mutant MspA monomer comprises one or more of the following mutations of SEQ ID NO:1: L88W, I105W, N108W, D118R, D134R, A138P, and E139A/K.

28. The method of claim 1 , wherein the mutant MspA monomer comprises mutations at amino acid positions 118, 134, and 139 of SEQ ID NO:1.

29. The method of claim 1 , wherein the mutant MspA monomer comprises one or more mutations such that at least one of amino acid positions 118, 134, and 139 of SEQ ID NO:1 contains an amino acid with positive charge.

30. The method of claim 1 , wherein the mutant MspA monomer comprises a mutation such that amino acid position 118 of SEQ ID NO:1 contains an amino acid with positive charge.

31. The method of claim 1 , wherein the mutant MspA monomer comprises mutations such that amino acid positions 118, 134, and 139 of SEQ ID NO:1 contain amino acids with positive charge.

32. The method of claim 1 , wherein the mutant MspA monomer comprises the following mutation of SEQ ID NO:1: D118R.

33. The method of claim 1 , wherein the mutant MspA monomer comprises the following mutation of SEQ ID NO:1: E139K.

34. The method of claim 1 , wherein the mutant MspA monomer comprises the following mutation of SEQ ID NO:1: D134R.

35. The method of claim 13 , wherein the mutant Msp

Assignees

• Univ Washington
• Uab Res Found

Inventors

• Gundlach Jens H
• Niederweis Michael
• Butler Thomas Z
• Pavlenok Mikhail
• Troll Mark A
• Sukumaran Suja

Classifications

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C07K14/35 Primary

  from Mycobacteriaceae (F) · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• A61K39/04

  Mycobacterium, e.g. Mycobacterium tuberculosis · CPC title

• G01N27/44704

  Details; Accessories · CPC title