Transmembrane pore consisting of two CsgG pores

1 . A transmembrane double pore forming a channel through a membrane, comprising a first CsgG pore, or a homologue thereof, whose tail is associated with the tail of a second CsgG pore, or a homologue thereof, wherein:

(i) the first CsgG pore, or homologue thereof, is a homooligomer and the second CsgG pore, or homologue thereof, is a homooligomer, and the first CsgG pore, or homologue thereof, comprises monomers that have a different amino acid sequence from the monomers of which the second CsgG pore, or homologue thereof, is comprised;

(ii) the first CsgG pore, or homologue thereof, is a homooligomer and the second CsgG pore, or homologue thereof, is a homooligomer, and the first CsgG pore, or homologue thereof, and/or the second CsgG pore, or homologue thereof is not a wild-type pore;

(iii) the first CsgG pore, or homologue thereof, is a heterooligomer and the second CsgG pore, or homologue thereof, is a homooligomer;

(iv) the first CsgG pore, or homologue thereof, is a homooligomer and the second CsgG pore, or homologue thereof, is a heterooligomer; or

(v) the first CsgG pore, or homologue thereof, is a heterooligomer and the second CsgG pore, or homologue thereof, is a heterooligomer, and

wherein at least one residue at the interface between the first CsgG pore, or homologue thereof, and the second CsgG pore, or homologue thereof, is bulkier than the residue present at the corresponding position in a wild type CsgG pore monomer.

2 . The transmembrane double pore according to claim 1 , wherein the first CsgG pore, or homologue thereof, and the second CsgG pore, or homologue thereof are identical.

3 . The transmembrane double pore according to claim 1 , wherein the first CsgG pore, or homologue thereof, and the second CsgG pore, or homologue thereof, are different.

4 . The transmembrane double pore according to claim 1 , wherein the first CsgG pore, or homologue thereof, is attached to the second CsgG pore, or homologue thereof, by hydrophobic interactions and/or by one or more disulphide bond.

5 . The transmembrane double pore according to claim 4 , wherein at least one monomer in the first CsgG pore, or homologue thereof, and/or at least one monomer in the second CsgG pore, or homologue thereof, comprises at least one cysteine residue at the interface between the first and second pores, which cysteine residue is not present in the wild type CsgG monomer, or the wild type CsgG homologue monomer.

6 . The transmembrane double pore according to claim 4 , wherein at least one monomer in the first CsgG pore, or homologue thereof, and/or at least one monomer in the second CsgG pore, or homologue thereof, comprises at least one residue at the interface between the first and second pores, which residue is more hydrophobic than the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer.

7 . The transmembrane double pore according to claim 1 , wherein at least one monomer in the first CsgG pore, or homologue thereof, and/or at least one monomer in the second CsgG pore, or homologue thereof, comprises at least one residue in the barrel region of the pore, which residue has less negative charge than the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer.

8 . The transmembrane double pore according to claim 1 , wherein at least one monomer in the first CsgG pore, or homologue thereof, comprises at least one residue in the constriction of the barrel region of the first pore, which residue decreases, maintains or increases the length of the constriction compared to the wild type CsgG pore, or the wild type CsgG homologue pore, and/or at least one monomer in the second CsgG pore, or homologue thereof, comprises at least one residue in the constriction of the barrel region of the second pore, which residue decreases, maintains or increases the length of the constriction compared to the wild type CsgG pore, or the wild type CsgG homologue pore.

9 . The transmembrane double pore according to claim 8 , wherein the at least one residue in the constriction of the barrel region of the pore is a residue that is additional to the residues present in the constriction of the wild type CsgG pore, or the wild type CsgG homologue pore.

10 . The transmembrane double pore according to claim 9 , wherein the at least one residue is S, G, SG, SGG, SGS, GS, GSS and/or GSG.

11 . A method for determining the presence, absence or one or more characteristics of a target analyte, comprising:

(a) contacting the target analyte with the transmembrane double pore according to claim 1 such that the target analyte moves with respect to the transmembrane double pore; and

(b) taking one or more measurements as the analyte moves with respect to the transmembrane double pore and thereby determining the presence, absence or one or more characteristics of the analyte.

12 . A kit for characterising a target analyte comprising (a) the transmembrane double pore according to claim 1 and (b) the components of a membrane.

13 . An apparatus for characterising target analytes in a sample, comprising an array of transmembrane double pores according to claim 1 in a plurality of membranes.

14 . The transmembrane double pore of claim 1 , the at least one residue at the interface between the first CsgG pore, or homologue thereof, and the second CsgG pore, or homologue thereof, is at a position corresponding to A98, A99, T104, V105, L113, Q114 or S115 of SEQ ID NO: 2.

15 . The transmembrane double pore of claim 14 , wherein the at least one residue at the interface between the first CsgG pore, or homologue thereof, and the second CsgG pore, or homologue thereof is:

(i) I, L, V, M, F, W, Y, N, Q, S or T where the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer is A;

(ii) L, M, F, W, Y, N, Q, R, D or E where the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer is T;

(iii) I, L, M, F, W, Y, N, Q where the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer is V;

(iv) M, F, W, Y, N, Q, R, D or E where the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer is L;

(v) F, W, Y where the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer is Q; and/or

(vi) M, F, W, Y, N, Q, E or R where the residue present at the corresponding position in the wild type CsgG monomer, or the wild type CsgG homologue monomer is S.