MSP nanopores and related methods

Patent metadata

Field	Value
Publication number	US-10870883-B2
Application number	US-201815996084-A
Country	US
Kind code	B2
Filing date	Jun 1, 2018
Priority date	Sep 22, 2008
Publication date	Dec 22, 2020
Grant date	Dec 22, 2020

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 Mycobacterium smegmatis porin A (MspA) comprising an MspA monomer, wherein the MspA monomer comprises a variant of the sequence in SEQ ID NO:1, and wherein the variant comprises (i) mutations resulting in amino acids without a negative charge at positions 90 and 91 and (ii) one or more mutations adding a positively charged amino acid to a vestibule or entrance of the MspA.

2. The MspA of claim 1 , wherein the variant comprises amino acids with a neutral charge at positions 90 and 91.

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

4. The MspA of claim 3 , wherein the variant comprises one or more of the following:
asparagine, glutamine, or tyrosine at position 90;
asparagine, glutamine, or tyrosine at position 91; and
asparagine or glutamine at position 93.

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

6. The MspA of claim 5 , wherein the variant comprises arginine at position 118.

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

8. The MspA of claim 7 , wherein the variant comprises arginine at position 134.

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

10. The MspA of claim 9 , wherein the variant comprises lysine at position 139.

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

12. The MspA of claim 11 , wherein the variant comprises an amino acid with a positive charge at one or more of positions 88, 105, 108, 126, and 138.

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

14. The MspA of claim 1 , wherein the MspA further comprises a second MspA monomer which is the same as or different from the MspA monomer, and wherein the MspA monomer is connected to the second MspA monomer by a linker with one or more amino acids.

15. A system comprising a Mycobacterium smegmatis porin (Msp) nanopore having a vestibule and a constriction zone that define a tunnel, wherein the tunnel is positioned between a first conductive liquid medium and a second conductive liquid medium allowing liquid communication between the first and second conductive liquid media, wherein the system is operative to detect an analyte when the system is subjected to an electric field sufficient to translocate the analyte from one conductive liquid medium to the other, and wherein the Msp is a mutant comprising at least a first mutant MspA monomer.

16. The system of claim 15 , wherein the first conductive liquid medium comprises the analyte.

17. The system of claim 15 , wherein the system is operative to detect a property of the analyte.

18. The system of claim 15 , wherein the tunnel is positioned in a lipid bilayer between the first conductive liquid medium and the second conductive liquid medium.

19. The system of claim 15 , wherein the first mutant MspA monomer comprises a variant of the sequence SEQ ID NO:1, and wherein the variant comprises (i) mutations resulting in amino acids without a negative charge at positions 90 and 91 and (ii) one or more mutations adding a positively charged amino acid to a vestibule or entrance of the MspA.

20. The system of claim 15 , wherein the variant comprises amino acids with a neutral charge at positions 90 and 91.

21. The system of claim 19 , wherein the variant comprises one or more of the following: asparagine, glutamine, or tyrosine at position 90; asparagine, glutamine, or tyrosine at position 91; and asparagine or glutamine at position 93.

22. The system of claim 19 , wherein the variant comprises an amino acid with a positive charge at one or more of positions 118, 134, and 139.

23. The system of claim 19 , wherein the variant comprises an amino acid with a positive charge at one or more of positions 88, 105, 108, 126, and 138.

24. The system of claim 19 , wherein the variant comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO:1.

25. The system of claim 15 , wherein the Msp further comprises a molecular motor, wherein the molecular motor is capable of moving an analyte into or through the tunnel with an average translocation velocity that is less than the average translocation velocity at which the analyte electrophoretically translocates into or through the tunnel in the absence of the molecular motor.

26. The system of claim 25 , wherein the molecular motor is selected from one of an enzyme, a polymerase, an exonuclease, a DNA binding protein, or a Klenow fragment.

27. The system of claim 15 , wherein the system comprises a plurality of nanopores.

28. The system of claim 27 , wherein the system comprises two different nanopores.

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

• G01N27/44791

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

• C12N13/00

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

• C07K14/35

  from Mycobacteriaceae (F) · CPC title

• A61K39/04

  Mycobacterium, e.g. Mycobacterium tuberculosis · CPC title