Mutant csgg pores
AssigneesOxford Nanopore Technologies PLC, Vib Vzw, Vrije Universiteit Brussel
InventorsStefan Howorka, Han Remaut, Lakmal Jayasinghe, Elizabeth Jayne Wallace, James Anthony Clarke, Richard George Hambley, Jonathan Bankes Pugh
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Abstract
The invention relates to mutant forms of the outer membrane located lipoprotein CsgG, in particular, modifications at one or more of positions Tyr51; Asn55; and Phe56. The invention also relates to analyte detection and characterisation using said mutant CsgG.
Claims76
At least one modified CsgG monomer wherein the monomer comprises a modification at one or more of positions Tyr51 ;
- Asn55;
- and Phe56.
A modified CsgG biological pore comprising at least one CsgG monomer, wherein the modified CsgG biological pore has no more than one channel constriction with a diameter in the range from 0.5 nm to 1.5 nm.
A biosensor comprising:
- a) an insulating layer;
- b) a CsgG biological pore within the insulating layer;
- and
- c) apparatus for measuring an electrical current through the biological pore.
Use of a CsgG biological pore for biological sensing applications, wherein the biological sensing application is analyte detection or nucleic acid sequencing.
A method for molecular sensing comprising:
- a) providing a CsgG biological pore formed of at least one CsgG monomer within an insulating layer;
- b) allowing the CsgG biological pore to interact with a test substrate;
- and
- c) taking one or more measurements during the interaction.
A mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO:
- 390, wherein the variant comprises a mutation at one or more of positions Y51, N55 and F56.
A mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO:
- 390, wherein the variant comprises one or more of the following:
- (i) one or more mutations at the following positions N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192;
- (ii) mutations at Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56;
- (iii) Q42R or Q42K;
- (iv) K49R;
- (v) N102R, N102F, N102Y or N102W;
- (vi) D149N, D149Q or D149R;
- (vii) E185N, E185Q or E185R;
- (viii) D195N, D195Q or D195R;
- (ix) E201N, E201Q or E201 R;
- (x) E203N, E203Q or E203R;
- and (xi) deletion of one or more of the following positions F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57.
A method for determining the presence, absence or one or more characteristics of a target analyte, comprising:
- a. contacting the target analyte with a CsgG pore or a mutant thereof such that the target analyte moves with respect to the pore;
- and
- b. taking one or more measurements as the analyte moves with respect to the pore and thereby determining the presence, absence or one or more characteristics of the analyte.
A method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a CsgG pore or a mutant thereof and a polynucleotide binding protein and thereby forming a sensor for characterising the target polynucleotide.
A sensor for characterising a target polynucleotide, comprising a complex between a CsgG pore or a mutant thereof and a polynucleotide binding protein.
Use of a CsgG pore or a mutant thereof to determine the presence, absence or one or more characteristics of a target analyte.
A kit for characterising a target analyte comprising (a) a CsgG pore or a mutant thereof and (b) the components of a membrane.
An apparatus for characterising target analytes in a sample, comprising (a) a plurality of a CsgG pores or mutants thereof and (b) a plurality of membranes.
A method of characterising a target polynucleotide, comprising:
- a) contacting the polynucleotide with a CsgG pore or a mutant thereof, a polymerase and labelled nucleotides such that phosphate labelled species are sequentially added to the target polynucleotide by the polymerase, wherein the phosphate species contain a label specific for each nucleotide;
- and
- b) detecting the phosphate labelled species using the pore and thereby characterising the polynucleotide.
Dependencies are inferred from the claim text.