MSP Nanopores and Related Methods

Patent metadata

Field	Value
Publication number	US-2016137703-A1
Application number	US-201614993952-A
Country	US
Kind code	A1
Filing date	Jan 12, 2016
Priority date	Sep 22, 2008
Publication date	May 19, 2016
Grant date	—

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

1 . (canceled)

2 . A Mycobacterium smegmatis porin A (Msp A) comprising an Msp A monomer, wherein the Msp A monomer comprises a variant of the sequence in SEQ ID NO: 1, and wherein the variant comprises (i) amino acids with a neutral charge at positions 90 and 91 and (ii) an amino acid with a positive charge at one or more of amino acid positions 118, 134, and 139.

3 . The Msp A of claim 2 , wherein the variant comprises an amino acid with a positive charge at position 118.

4 . The Msp A of claim 2 , wherein the variant comprises an amino acid with a positive charge at positions 118, 134, and 139.

5 . The Msp A of claim 2 , wherein the variant comprises arginine at position 118.

6 . The Msp A of claim 2 , wherein the variant comprises lysine at position 139.

7 . The Msp A of claim 2 , wherein the variant comprises arginine at position 134.

8 . The Msp A of claim 2 , wherein the variant comprises arginine at position 118 and lysine at position 139.

9 . The Msp A of claim 4 , wherein the variant comprises arginine at position 118, arginine at position 134, and lysine at position 139.

10 . The Msp A of claim 2 , wherein the variant comprises asparagine or glutamine at position 90 and asparagine or glutamine at position 91.

11 . The Msp A of claim 2 , wherein the variant comprises asparagine at positions 90 and 91.

12 . The Msp A of claim 2 , wherein the variant comprises glutamine at positions 90 and 91.

13 . The Msp A of claim 2 , wherein the variant comprises an amino acid substitution at position 88.

14 . The Msp A of claim 4 , wherein the variant comprises an amino acid substitution at position 88.

15 . The Msp A of claim 10 , wherein the variant comprises an amino acid substitution at position 88.

16 . The Msp A of claim 2 , wherein the variant comprises an amino acid substitution at position 105.

17 . The Msp A of claim 2 , wherein the variant comprises an amino acid substitution at position 108.

18 . The Msp A of claim 2 , wherein the variant comprises an amino acid with a positive charge at position 126.

19 . The Msp A of claim 2 , wherein the variant comprises aspartic acid at position 93.

20 . The Msp A of claim 4 , wherein the variant comprises aspartic acid at position 93.

21 . The Msp A of claim 10 , wherein the variant comprises aspartic acid at position 93.

22 . The Msp A of claim 2 , wherein the variant comprises asparagine at positions 90, 91, and 93, arginine at positions 118 and 134, and lysine at position 139.

23 . The Msp A of claim 2 , wherein the Msp A comprises a property of exhibiting blockades when ssDNA is present in 1 M KCl on a cis side of the Msp A at an applied voltage of about 80 mV.

24 . The Msp A of claim 2 , wherein the MspA further comprises a second MspA monomer, and wherein the MspA monomer is connected to the second MspA monomer by one or more amino acid linkers.

25 . A Mycobacterium smegmatis porin A (Msp A) comprising an Msp A monomer, wherein the Msp A monomer comprises a variant of the sequence in SEQ ID NO: 1, and wherein the variant comprises (i) amino acids with a neutral charge at positions 90 and 91 and (ii) one or more amino acid substitutions adding a positively charged amino acid to a vestibule of the Msp A.

26 . The Msp A of claim 25 , wherein the variant comprises aspartic acid at position 93.

27 . The Msp A of claim 26 , wherein the variant comprises asparagine or glutamine at position 90 and asparagine or glutamine at position 91.

28 . The Msp A of claim 26 , wherein the variant comprises asparagine at positions 90 and 91.

29 . The Msp A of claim 25 , wherein the variant comprises an amino acid substitution at position 88.

30 . The Msp A of claim 25 , wherein the Msp A comprises a property of exhibiting blockades when ssDNA is present in 1 M KCl on a cis side of the Msp A at an applied voltage of about 80 mV.

31 . The Msp A of claim 25 , wherein the MspA further comprises a second MspA monomer, and wherein the MspA monomer is connected to the second MspA monomer by one or more amino acid linkers.

Assignees

• Univ Washington
• Uab Research Foundation

Inventors

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

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07K14/35 Primary

  from Mycobacteriaceae (F) · CPC title

• G01N27/44704

  Details; Accessories · 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