MSP nanopores and related methods
Inventors
GUNDLACH, Jens H. • Niederweis, Michael • BUTLER, Thomas Z. • PAVLENOK, Mikhail • Troll, Mark A. • Sukumaran, Suja • Hille, Bertil
Assignees
WASHINGTON, University of • University of Washington • UAB Research Foundation
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Publication Number
US-8673550-B2
Publication Date
2014-03-18
Expiration 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.
Core Innovation
The disclosure relates to Mycobacterium smegmatis porin (Msp) nanopore technology using a porin having a vestibule and a constriction zone that define a tunnel. Analytes are detected by subjecting the Msp tunnel, positioned between a first conductive liquid medium and a second conductive liquid medium in liquid communication, to an electric field sufficient to translocate the analyte between the conductive liquid media. The disclosure measures an ion current to detect analytes based on an ion current reduction relative to an Msp without an analyte present.
Detection is based on measuring an ion current wherein a 5% or more reduction in the ion current for at least 1.0 μs compared to an ion current level for the Msp without an analyte present indicates the presence of the analyte in the first medium. The disclosure further describes current pattern comparisons and current blockade comparisons to reference current patterns obtained using a known analyte. It also describes operability for analyte detection when the system is subjected to an electric field sufficient to translocate the analyte.
The disclosure also provides tunnel conductance modification and engineered Msp structures by altering amino acids in the vestibule or constriction zone of a wild-type Msp to define a conductive tunnel distinct from the tunnel defined by the wild-type Msp. It further describes mutant MspA monomers with mutations at specified positions, including mutations at position 93 together with mutations at position 90, position 91, or both positions 90 and 91. In addition, it describes mutant single-chain Msp constructs encoded by nucleic acid sequences that link multiple Msp monomer sequences using an amino acid linker sequence, with at least one monomer containing amino acid deletion, substitution, or addition in the periplasmic loop, vestibule, or constriction zone.
Claims Coverage
The partial content contains multiple independent claims covering five inventive features: analyte detection using an Msp tunnel and ion current reduction under an applied electric field, conductance modification by amino-acid changes in the vestibule or constriction zone, an Msp-based detection system, mutant MspA monomers with specified position mutations, and mutant single-chain Msp nucleic-acid constructs. The independent claim set identifies the main inventive features as a tunnel defined by a vestibule and constriction zone, electric-field-driven analyte translocation between conductive liquid media, ion-current-based detection, and region-targeted amino-acid engineering of Msp/MspA and single-chain architectures.
Electric-field analyte detection using Msp tunnel ion-current reduction
Applying an electric field sufficient to translocate an analyte from a first conductive liquid medium to a second conductive liquid medium in liquid communication through an Msp having a vestibule and a constriction zone that define a tunnel; and measuring an ion current, wherein a 5% or more reduction in the ion current for at least 1.0 μs compared to an ion current level for the Msp without an analyte present indicates the presence of the analyte in the first medium.
Conductance modification by vestibule or constriction-zone amino-acid change
Modifying conductance through the tunnel of an Msp by removing, adding, or replacing at least one amino acid in the vestibule or the constriction zone of a wild-type Msp to define a conductive tunnel that is distinct from the tunnel defined by the wild-type Msp.
Msp detection system between conductive liquid media with electric-field translocation
A system comprising an Msp 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 at least one conductive liquid medium comprises an analyte, and wherein the system is operative to detect the analyte when the system is subjected to an electric field sufficient to translocate the analyte from one conductive liquid medium to the other.
Mutant MspA monomer with position 93 and position 90/91 mutations
A mutant MspA comprising a vestibule and a constriction zone that define a tunnel, and at least a first mutant MspA monomer comprising a mutation at position 93 and a mutation at position 90, position 91, or both positions 90 and 91.
Mutant single-chain Msp nucleic-acid construct with linker and region-targeted mutations
A mutant single-chain Msp encoded by a nucleic acid sequence comprising a first and second nucleotide sequence encoding a first Msp monomer sequence and a second Msp monomer sequence, and a third nucleotide sequence encoding an amino acid linker sequence, wherein at least one of the first and second Msp monomers comprises at least one amino acid deletion, substitution or addition in the sequence for the periplasmic loop, vestibule, or constriction zone of the Msp.
Across the independent claims, the patent coverage centers on detecting analytes by electric-field translocation through an Msp tunnel defined by a vestibule and constriction zone and identifying the analyte using ion current reduction and related current-pattern or blockade comparisons. Additional coverage specifies engineering Msp/MspA tunnel conductance and structure by region-targeted amino-acid changes, mutant MspA monomers with mutations at position 90 and/or 91 and position 93, and mutant single-chain Msp nucleic-acid architectures encoding multiple linked monomer segments with modifications in the periplasmic loop, vestibule, or constriction zone.
Stated Advantages
Detects the presence of an analyte by indicating the presence of the analyte when a 5% or more reduction in ion current for at least 1.0 μs occurs relative to ion current for the Msp without an analyte present.
Defines a conductive tunnel distinct from the tunnel defined by the wild-type Msp by removing, adding, or replacing amino acids in the vestibule or constriction zone.
Documented Applications
Detecting the presence of an analyte using an Msp tunnel in a system positioned between conductive liquid media and operated under an electric field sufficient to translocate the analyte.
Modifying tunnel conductance of an Msp by changing amino acids in the vestibule or constriction zone of a wild-type Msp.
Using mutant MspA monomers with specified mutations, including at position 93 and at position 90 and/or 91, and mutant single-chain Msp constructs with a linker and region-targeted mutations in the periplasmic loop, vestibule, or constriction zone.
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