Mutant pore

Patent metadata

Field	Value
Publication number	US-10975428-B2
Application number	US-201716081894-A
Country	US
Kind code	B2
Filing date	Mar 2, 2017
Priority date	Mar 2, 2016
Publication date	Apr 13, 2021
Grant date	Apr 13, 2021

Abstract

The invention relates to mutant forms of CsgG. The invention also relates to analyte detection and characterisation using CsgG.

First claim

The invention claimed is:

1. A method for determining the presence, absence, or one or more characteristics of a target analyte, comprising:
(a) contacting the target analyte with a pore such that the target analyte moves with respect to the pore, wherein the pore is a homo-oligomer that comprises a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO: 2 which comprises: R97W; R93W; R93Y and R97Y; F191T; deletion of V105, A106 and I107; and/or deletion of one or more of positions R192, F193, I194, D195, Y196, Q197, R198, L199, L200 and E201; 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.

2. The method of claim 1 , wherein the variant comprises R97W.

3. The method of claim 1 , wherein the variant comprises deletion of F193, I194, D195, Y196, Q197, R198 and L199 or deletion of D195, Y196, Q197, R198 and L199.

4. The method of claim 1 , 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 or K94Q; (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 E201R; (x) E203N, E203Q or E203R; (xi) deletion of one or more of the following positions F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57; and (xii) one or more mutations at the following positions L90, N91, I95, A99, Q114.

5. The method of claim 1 , wherein the variant comprises (a) a mutation at Y51 and/or F56, and/or (b) a mutation at R192, and/or (c) a mutation at T150.

6. The method of claim 5 , wherein the mutation at Y51 is Y51A, and/or the mutation at F56 is F56Q, and/or the mutation at R192 is R192D.

7. The method of claim 1 , wherein the pore comprises nine identical mutant CsgG monomers.

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Jayasinghe Lakmal
• Wallace Elizabeth Jayne

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07K14/245 Primary

  Escherichia (G) · CPC title

• G01N33/48721

  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) · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title