Msp nanopores and related methods

Patent metadata

Field	Value
Publication number	US-9534024-B2
Application number	US-201414216349-A
Country	US
Kind code	B2
Filing date	Mar 17, 2014
Priority date	Sep 22, 2008
Publication date	Jan 3, 2017
Grant date	Jan 3, 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 invention claimed is:

1. A nucleic acid sequence encoding a single-chain Msp, wherein the nucleic acid sequence comprises:
(a) a first and second nucleotide sequence, wherein the first nucleotide sequence encodes a first Msp monomer sequence and the second nucleotide sequence encodes a second Msp monomer sequence; and
(b) a third nucleotide sequence encoding an amino acid linker sequence that links the first Msp monomer sequence and the second Msp monomer sequence.

2. The nucleic acid sequence of claim 1 , wherein the first and second Msp monomer sequences are independently selected from the group consisting of a wild-type MspA monomer, a mutant MspA monomer, a wild-type MspA paralog or homolog monomer, and a mutant MspA paralog or homolog monomer.

3. The nucleic acid sequence of claim 1 , wherein the first Msp monomer sequence comprises one or more of the mutations of SEQ ID NO:1 selected from the group consisting of an A to P substitution at amino acid 138, an E to A or K substitution at amino acid 139, a D to K or R or Q substitution at amino acid 90; a D to N or Q substitution at amino acid 91, a D to N substitution at amino acid 93, an L to W substitution at amino acid 88, an I to W substitution at amino acid 105, an N to W substitution at amino acid 108, a D to R substitution at amino acid 118, and a D to R substitution at amino acid 134.

4. The nucleic acid sequence of claim 3 , wherein the mutant MspA monomer comprises an A to P substitution at amino acid 138 of SEQ ID NO:1, an E to A substitution at amino acid 139 of SEQ ID NO:1, or a combination thereof.

5. The nucleic sequence of claim 3 , wherein the mutant MspA monomer comprises a D to K or R substitution at amino acid 90 of SEQ ID NO:1, a D to N substitution at amino acid 91 of SEQ ID NO:1, a D to N substitution at amino acid 93 of SEQ ID NO:1, or any combination thereof.

6. The nucleic acid sequence of claim 3 , wherein the mutant MspA monomer comprises a D to Q substitution at amino acid 90 of SEQ ID NO:1, a D to Q substitution at amino acid 91 of SEQ ID NO:1, a D to N substitution at amino acid 93 of SEQ ID NO:1, or any combination thereof.

7. The nucleic acid sequence of claim 3 , wherein the mutant MspA monomer comprises an L to W substitution at amino acid 88 of SEQ ID NO:1, an I to W substitution at amino acid 105 of SEQ ID NO:1, a D to Q substitution at amino acid 91 of SEQ ID NO:1, a D to N substitution at amino acid 93 of SEQ ID NO:1, or any combination thereof.

8. The nucleic acid sequence of claim 3 , wherein the mutant MspA monomer comprises an I to W substitution at amino acid 105 of SEQ ID NO:1, an N to W substitution at amino acid 108 of SEQ ID NO:1, or a combination thereof.

9. The nucleic acid sequence of claim 3 , wherein the mutant MspA monomer comprises a D to R substitution at amino acid 118 of SEQ ID NO:1, an E to K substitution at amino acid 139 of SEQ ID NO:1, a D to R substitution at amino acid 134 of SEQ ID NO:1, or any combination thereof.

10. The nucleic acid sequence of claim 1 , wherein the first Msp monomer sequence comprises SEQ ID NO:1.

11. The nucleic acid sequence of claim 1 , wherein the second Msp monomer sequence comprises SEQ ID NO:2.

12. The nucleic acid sequence of claim 1 , wherein the first Msp monomer sequence comprises SEQ ID NO:1 and the second Msp monomer sequence comprises SEQ ID NO:2.

13. A vector comprising the nucleic acid sequence of claim 1 .

14. A nucleic acid sequence encoding a single-chain Msp, wherein the nucleic acid sequence comprises:
(a) a first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequence, wherein the first, second, third, fourth, fifth, sixth, seventh, and eighth nucleotide sequences are arranged consecutively in the nucleic acid sequence and encode a first, second, third, fourth, fifth, sixth, seventh, and eighth Msp monomer sequence, respectively; and
(b) at least one ninth nucleotide sequence encoding an amino acid linker sequence that links any two consecutive Msp monomer sequences.

15. The nucleic acid sequence of claim 14 , wherein the first and second Msp monomer sequences are independently selected from the group consisting of a wild-type Msp monomer, a mutant Msp monomer, a wild-type MspA paralog or homolog monomer, and a mutant MspA paralog or homolog monomer.

16. The nucleic acid sequence of claim 15 , wherein at least one Msp monomer comprises a mutant MspA monomer.

17. The nucleic acid sequence of claim 16 , wherein the mutant Msp monomer sequence comprises one or more of the mutations of SEQ ID NO:1 selected from the group consisting of an A to P substitution at amino acid 138, an E to A or K substitution at amino acid 139, a D to K or R or Q substitution at amino acid 90; a D to N or Q substitution at amino acid 91, a D to N substitution at amino acid 93, an L to W substitution at amino acid 88, an I to W substitution at amino acid 105, an N to W substitution at amino acid 108, a D to R substitution at amino acid 118, and a D to R substitution at amino acid 134.

18. The nucleic acid sequence of claim 17 , wherein the mutant MspA monomer comprises an A to P substitution at amino acid 138 of SEQ ID NO:1, an E to A substitution at amino acid 139 of SEQ ID NO:1, or a combination thereof.

19. The nucleic sequence of claim 17 , wherein the mutant MspA monomer comprises a D to K or R substitution at amino acid 90 of SEQ ID NO:1, a D to N substitution at amino acid 91 of SEQ ID NO:1, a D to N substitution at amino acid 93 of SEQ ID NO:1, or any combination thereof.

20. The nucleic acid sequence of claim 17 , wherein the mutant MspA monomer comprises a D to Q substitution at amino acid 90 of SEQ ID NO:1, a D to Q substitution at amino acid 91 of SEQ ID NO:1, a D to N substitution at amino acid 93 of SEQ ID NO:1, or any combination thereof.

21. The nucleic acid sequence of claim 17 , wherein the mutant MspA monomer comprises an L to W substitution at amino acid 88 of SEQ ID NO:1, an I to W substitution at amino acid 105 of SEQ ID NO:1, a D to Q substitution at amino acid 91 of SEQ ID NO:1, a D to N substitution at amino acid 93 of SEQ ID NO:1, or any combination thereof.

22. The nucleic acid sequence of claim 17 , wherein the mutant MspA monomer comprises an I to W substitution at amino acid 105 of SEQ ID NO:1, an N to W substitution at amino acid 108 of SEQ ID NO:1, or a combination thereof.

23. The nucleic acid sequence of claim 17 , wherein the mutant MspA monomer comprises a D to R substitution at amino acid 118 of SEQ ID NO:1, an E to K substitution at amino acid 139 of SEQ ID NO:1, a D to R substitution at amino acid 134 of SEQ ID NO:1, or any combination thereof.

24. The nucleic acid sequence of claim 14 , wherein each Msp monomer sequence comprises SEQ ID NO:1.

25. The nucleic acid sequence of claim 14 , wherein at least one Msp monomer sequence comprises SEQ ID NO:1.

26. The nucleic acid sequence of claim 14 , wherein at least one Msp monomer sequence comprises a wild-type MspA paralog or mutant thereof, wherein the MspA paralog or mutant thereof is a wild-type MspB monomer or a mutant thereof.

27. The nucleic acid sequence of claim 25 , wherein at least one Msp monomer sequence comprises SEQ ID NO:2.

28. The nucleic acid sequence of claim 14 , wherein at least one Msp monomer sequence comprises a wild-type MspA monomer or a mutant thereof and at least one Msp monomer sequence comprises a wil

Assignees

• Univ Washington
• Uab Res Found

Inventors

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

Classifications

• G01N33/6872

  Intracellular protein regulatory factors and their receptors, e.g. including ion channels · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• 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

• G01N24/00

  Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects · CPC title