# US 10,870,883 B2 · MSP nanopores and related methods

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- Number: US 10,870,883 B2
- Document ID: US10870883B2
- Status: Granted
- Kind: B2
- Application number: 15/996,084
- Filed: 2018-06-01
- Published: 2019-02-28
- Granted: 2020-12-22
- Earliest claimed: 2008-09-22
- Assignees: University of Washington; The UAB Research Foundation
- Applicants: University of Washington; The UAB Research Foundation
- Inventors: Jens H. Gundlach; Michael Niederweis; Thomas Z. Butler; Mikhail Pavlenok; Mark A. Troll; Suja Sukumaran
- Web page: https://patentgrep.com/patent/US10870883B2

## CPC

- C12Q 1/6869: Methods for sequencing
- A61K 39/04: Mycobacterium, e.g. Mycobacterium tuberculosis
- C07K 14/35: from Mycobacteriaceae (F)
- C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
- G01N 27/44704: {Details; Accessories}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- 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)}
- G01N 33/6872: {Intracellular protein regulatory factors and their receptors, e.g. including ion channels}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- G01N 24/00: Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 3)

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.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 7)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 9)

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

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 11)

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.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 1)

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.

### Claim 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.

### Claim 16 (depends on 15)

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

### Claim 17 (depends on 15)

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

### Claim 18 (depends on 15)

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.

### Claim 19 (depends on 15)

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.

### Claim 20 (depends on 15)

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

### Claim 21 (depends on 19)

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.

### Claim 22 (depends on 19)

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.

### Claim 23 (depends on 19)

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.

### Claim 24 (depends on 19)

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

### Claim 25 (depends on 15)

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.

### Claim 26 (depends on 25)

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.

### Claim 27 (depends on 15)

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

### Claim 28 (depends on 27)

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