MSP NANOPORES AND RELATED METHODS

• US 20140309402A1
• Filed: 03/17/2014
• Published: 10/16/2014
• Est. Priority Date: 09/22/2008
• Status: Active Grant

• Current Assignee: UAB Research Foundation (University of Alabama System), University of Washington
• Original Assignee: UAB Research Foundation (University of Alabama System), University of Washington
• Inventors: Gundlach, Jens H., Niederweis, Michael, Butler, Thomas Z., Pavlenok, Mikhail, Troll, Mark A., Sukumaran, Suja
• Granted Patent: US 9,170,230 B2
• Time in Patent Office: Days
• US Class Current: 530/350
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/04 Mycobacterium, e.g. Mycobacterium tuberculosis
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/31 Electrostatic interactions, e.g. use of cationic polymers in hybridisation reactions
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 24/00 Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/48721 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 Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

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

1. (canceled)

2. A mutant Mycobacterium smegmatis porin A (Msp A) 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, or 139.

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

4. The mutant Msp A monomer of claim 2, wherein the variant comprises the following mutations:

D90N, D91N, and D93N.

5. The mutant Msp A monomer of claim 2, wherein the variant comprises the following mutations:

D90N, D91N, D93N, D118R, D134R, and E139K.

6. The mutant Msp A monomer of claim 2, wherein the variant comprises the following mutations:

D90N/Q/Y, D91N/Q/Y, and D93N/Q.

7. The mutant Msp A monomer of claim 2, wherein the variant comprises the mutation D93N.

8. The mutant Msp A monomer of claim 2, wherein the variant comprises one or more of the following mutations:

L88W, I105W, N108W, D118R, D134R, A138P, and E139A/K.

9. The mutant Msp A monomer of claim 2, wherein the variant comprises a mutation at amino acid position 88.

10. The mutant Msp A monomer of claim 2, wherein the variant comprises a mutation at amino acid position 126.

11. The mutant Msp A monomer of claim 2, wherein the monomer is linked by an amino acid linker sequence to a second mutant Msp A monomer.

12. The mutant Msp A monomer of claim 11, wherein the amino acid linker sequence is 10 to 20 amino acids in length.

13. A mutant Mycobacterium smegmatis porin A (Msp A) 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.

14. The mutant Msp A monomer of claim 13, wherein the variant comprises one or more of the following mutations:

L88W, 1105W, N108W, D118R, D134R, A138P, and E139A/K.

15. The mutant Msp A monomer of claim 13, wherein the variant comprises one or more mutations at the following positions:

88, 105, 108, 118, 126, 134, or 139.

16. The mutant Msp A monomer of claim 13, wherein the variant consists of the following mutations:

D90N, D91N, D93N, optionally D118R, optionally D134R, and optionally E139K.

17. The mutant Msp A monomer of claim 13, wherein the variant comprises a mutation at amino acid position 88.

18. The mutant Msp A monomer of claim 13, wherein the variant comprises a mutation at amino acid position 126.

19. A mutant Mycobacterium smegmatis porin A (Msp A) monomer for nucleic acid sequencing, the mutant Msp A 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.

20. The mutant Msp A monomer of claim 19, wherein the variant has the following mutations:

D90N, D91N, and D93N.

21. The mutant Msp A monomer of claim 19, wherein the variant has the following mutations:

D90N, D91N, D93N, DI 18R, D134R, and E139K.