Mutant CsgG pores
Inventors
Howorka, Stefan • Remaut, Han • Jayasinghe, Lakmal • Wallace, Elizabeth Jayne • Clarke, James • Hambley, Richard George • Pugh, Jonathan Bankes
Assignees
Vlaams Instituut voor Biotechnologie VIB • Vrije Universiteit Brussel VUB • Oxford Nanopore Technologies PLC
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Publication Number
US-12275761-B2
Publication Date
2025-04-15
Expiration Date
Abstract
The invention relates to mutant forms of CsgG. The invention also related to analyte detection and characterisation using CsgG.
Core Innovation
The invention relates to transmembrane pores comprising at least one CsgG monomer, including mutant CsgG biological nanopores, chemically fused CsgG monomer constructs, and genetically fused constructs. The CsgG monomer variants are defined by specified mutations and/or deletions at selected positions, and the pore architecture includes nonameric, octameric, homo-oligomeric, hetero-oligomeric, and construct-containing pore formats.
The pore compositions are used as transmembrane nanopores in an insulating membrane such that a target analyte moves through the pore. Electrical current-based sensing and one or more electrical measurements are performed while the analyte translocates through the pore, including nanopore-based analyte sensing, characterisation, and high-resolution nucleotide discrimination.
For polynucleotide approaches, the target analyte is a target polynucleotide contacted with a polynucleotide binding protein that controls movement of the polynucleotide through the pore. The polynucleotide binding protein is described as a helicase or derived from a helicase, and the disclosure also includes nanopore sequencing approaches using applied potential and membrane formats for tethering polynucleotides to pore-containing membranes.
Claims Coverage
The claim coverage centers on one independent method claim for measuring a target analyte using a transmembrane pore comprising at least one CsgG monomer and obtaining electrical measurements during translocation, with dependent refinements that specify the CsgG monomer variant and optionally add polynucleotide-binding-protein control. Across the items, four inventive features are identified: CsgG pore translocation measurements, specified CsgG monomer variants, polynucleotide analytes with binding-protein-controlled movement, and helicase or helicase-derived binding proteins.
Electrical measurement of translocation through a CsgG pore
Contacting the target analyte with a transmembrane pore comprising at least one CsgG monomer such that the target analyte moves through the pore, and taking one or more electrical measurements as the target analyte moves through the pore.
Specified CsgG monomer variant for the pore
Defining the at least one CsgG monomer as a variant of SEQ ID NO: 390 containing specified mutations at selected positions and/or specified deletions at selected positions.
Polynucleotide analyte with binding-protein controlled movement
Modifying the method so that the target analyte is a target polynucleotide and contacting the polynucleotide with a polynucleotide binding protein that controls movement of the polynucleotide through the pore.
Helicase or helicase-derived polynucleotide binding protein
Defining the polynucleotide binding protein as a helicase or derived from a helicase.
The claims emphasize electrical measurements during analyte translocation through a transmembrane pore containing at least one CsgG monomer, including specifically defined CsgG monomer variants, and extend to target polynucleotides whose movement through the pore is controlled by a polynucleotide binding protein, optionally a helicase or helicase-derived protein.
Stated Advantages
Improved nucleotide discrimination, including increased current range and reduced current-state variance.
Fewer nucleotides contributing to the current signal.
Increased throughput/insertion.
Improved signal-to-noise ratio.
High-resolution nucleotide discrimination.
Documented Applications
Electrical current-based molecular sensing using CsgG nanopores and nucleic acid sequencing of DNA or RNA target polynucleotides.
Nanopore-based analyte sensing/characterisation, including nanopore sequencing approaches.
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