MSP nanopores and related methods

Patent metadata

Field	Value
Publication number	US-11634764-B2
Application number	US-202017121621-A
Country	US
Kind code	B2
Filing date	Dec 14, 2020
Priority date	Sep 22, 2008
Publication date	Apr 25, 2023
Grant date	Apr 25, 2023

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 nucleic acid comprising a sequence encoding a Mycobacterium smegmatis porin A (MspA) monomer, wherein the encoded MspA monomer comprises a variant amino acid sequence of SEQ ID NO:1, the variant amino acid sequence comprising:
(i) mutations resulting in amino acids with a neutral charge at each of positions 90 and 91, wherein the mutations at positions 90 and 91 are each individually selected from asparagine, glutamine, or tyrosine; and
(ii) one or more mutations replacing a negatively charged amino acid in SEQ ID NO:1 with a positively charged amino acid, thereby adding one or more positively charged amino acid to a vestibule or entrance of an MspA comprising the encoded MspA monomer.

2. The nucleic acid of claim 1 , wherein the variant amino acid sequence further comprises an amino acid with a neutral charge at position 93.

3. The nucleic acid of claim 2 , wherein the amino acid with the neutral charge at position 93 is selected from one of
asparagine or glutamine.

4. The nucleic acid of claim 1 , wherein the variant amino acid sequence comprises an amino acid with a positive charge at position 118.

5. The nucleic acid of claim 4 , wherein the variant amino acid sequence comprises arginine at position 118.

6. The nucleic acid of claim 1 , wherein the variant amino acid sequence comprises an amino acid with a positive charge at position 134.

7. The nucleic acid of claim 6 , wherein the variant amino acid sequence comprises arginine at position 134.

8. The nucleic acid of claim 1 , wherein the variant amino acid sequence comprises an amino acid with a positive charge at position 139.

9. The nucleic acid of claim 8 , wherein the variant amino acid sequence comprises lysine at position 139.

10. The nucleic acid of claim 1 , wherein the variant amino acid sequence further comprises an amino acid substitution at one or more of positions 88, 105, 108, 126, and 138.

11. The nucleic acid of claim 10 , wherein the variant amino acid sequence comprises one or more of the following substitutions: L88W, I105W, N108W, Q126R/W, and A138P.

12. The nucleic acid of claim 1 , wherein the variant amino acid sequence comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:1.

13. The nucleic acid of claim 1 , wherein the nucleic acid also encodes a second MspA monomer which is the same as or different from the MspA monomer, and wherein the encoded MspA monomer is connected to the second encoded MspA monomer by a linker with one or more amino acids.

14. A vector comprising the nucleic acid of claim 1 .

15. The vector of claim 14 , further comprising a promoter.

16. The vector of claim 15 , wherein the promoter is a constitutive promoter.

17. The vector of claim 15 , wherein the promoter is an inducible promoter.

18. A cell transfected with the vector of claim 14 .

19. The cell of claim 18 , wherein the cell is capable of expressing an Msp porin.

20. The nucleic acid of claim 1 , wherein the one or more mutations replacing a negatively charged amino acid with a positively charged amino acid in SEQ ID NO:1 are at positions selected from amino acid positions 118, 134, and 139 of SEQ ID NO:1.

21. A nucleic acid comprising a sequence encoding a Mycobacterium smegmatis porin A (MspA) monomer, wherein the encoded MspA monomer comprises a variant amino acid sequence of SEQ ID NO:1, and wherein the variant amino acid sequence comprises (i) mutations resulting in amino acids without a negative charge at positions 90 and 91 and (ii) one or more mutations replacing a negatively charged amino acid with a positively charged amino acid at one or more of amino acid positions 118, 134, and 139.

Assignees

• Univ Washington
• Uab Res Found

Inventors

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

Classifications

• A61K39/00

  Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• G01N24/00

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

• G01N27/44704

  Details; Accessories · 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