Mutant lysenin pores

Patent metadata

Field	Value
Publication number	US-11845780-B2
Application number	US-202017108536-A
Country	US
Kind code	B2
Filing date	Dec 1, 2020
Priority date	Apr 10, 2012
Publication date	Dec 19, 2023
Grant date	Dec 19, 2023

Abstract

The invention relates to mutant forms of lysenin. The invention also relates to analyte characterisation using lysenin.

First claim

The invention claimed is:

1. An apparatus for characterizing target polynucleotides in a sample comprising (a) a plurality of pores derived from lysenin, each lysenin pore comprising at least one mutant lysenin monomer comprising a barrel-forming region comprising the amino acid sequence as set forth in positions 44 to 126 of SEQ ID NO: 2 with one or more amino acid substitutions, wherein at least one of the one or more amino acid substitutions introduces a cysteine, lysine, or non-natural amino acid, and (b) a plurality of polynucleotide binding proteins.

2. The apparatus according to claim 1 , wherein the apparatus comprises:
(i) a sensor device that is capable of supporting the plurality of pores and being operable to perform polynucleotide characterisation using the pores and proteins;
(ii) at least one reservoir for holding material for performing the characterisation;
(iii) a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and
(iv) a plurality of containers for receiving respective samples, the fluidics system being configured to supply the samples selectively from the containers to the sensor device.

3. The apparatus of claim 1 , wherein the at least one of the cysteine, lysine, or non-natural amino acid is further attached to (i) a polyethylene glycol, a nucleic acid, a dye, a fluorophore, or a chromophore; (ii) a molecular adaptor; or (iii) a polynucleotide binding protein.

4. The apparatus of claim 1 , wherein the one or more amino acid substitutions (a) alter the steric effect of the monomer, (b) alter the net charge of the monomer, (c) alter the ability of the monomer to hydrogen bond with a polynucleotide, (d) introduce or remove chemical groups that interact through delocalized electron pi systems and/or (e) alter the structure of the monomer.

5. The apparatus of claim 4 , wherein (i) the one or more amino acid substitutions increase or decrease net positive charge; (ii) net positive charge is increased by introducing one or more positively charged amino acids and/or neutralising one or more negative charges; or (iii) one or more negative charges are neutralised by substituting one or more negatively charged amino acids with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids or by introducing one or more positive charged amino acids adjacent to one or more negatively charged amino acids.

6. The apparatus of claim 1 , wherein the one or more substitutions comprise substitution at one or more of the following positions of SEQ ID NO: 2: M44, N46, N48, E50, R52, H58, D68, F70, E71, S74, E76, S78, Y79, S80, H81, S82, E84, E85, S86, Q87, S89, M90, E92, E94, E97, E102, H103, T104, T106, R115, Q117, N119, D121 and D126.

7. The apparatus of claim 6 , wherein the amino acid(s) substituted into the one or more positions are selected from asparagine (N), serine (S), glutamine (Q), arginine (R), glycine (G), tyrosine (Y), aspartic acid (D), leucine (L), lysine (K) or alanine (A).

8. The apparatus of claim 1 , wherein the one or more substitutions comprise one or more of the following amino acid substitutions:
(a) serine (S) at position 44;
(b) serine (S) at position 46;
(c) serine (S) at position 48;
(d) serine (S) at position 52;
(e) serine (S) at position 58;
(f) serine (S) at position 68;
(g) serine (S) at position 70;
(h) serine (S) at position 71;
(i) serine (S) at position 76;
(j) serine (S) at position 79;
(k) serine (S) at position 81;
(l) serine (S), aspartic acid (D) or glutamine (Q) at position 84;
(m) serine (S) or lysine (K) at position 85;
(n) serine (S) at position 87;
(o) serine (S) at position 90;
(p) asparagine (N) or glutamine (Q) at position 92;
(q) serine (S) or asparagine (N) at position 94;
(r) serine (S) or asparagine (N) at position 97;
(s) serine (S) at position 102;
(t) serine (S) at position 103;
(u) asparagine (N) or serine (S) at position 121;
(v) serine (S) at position 50;
(w) asparagine (N) or serine (S) at position 94;
(x) asparagine (N) or serine (S) at position 97;
(y) serine (S) or asparagine (N) at position 121;
(z) asparagine (N) or glutamine (Q) or glycine (G) at position 126; and
(aa) serine (S) or asparagine (N) at position 128.

9. The apparatus of claim 8 , wherein the one or more substitutions comprises amino acid substitutions selected from the group consisting of:
i. one or more of E84D and E85K;
ii. one or more of E84Q, E85K, E92Q, E97S, D126G and E167A;
iii. one or more of E92N, E94N, E97N, D121N and D126N;
iv. one or more of E92N, E94N, E97N, D121N, D126N and E128N;
v. one or more of E76S, E84Q, E85K, E92Q, E97S, D126G and E167A;
vi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E50S;
vii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E71S;
viii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E94S;
ix. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E102S;
x. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E128S;
xi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E135S;
xii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D68S;
xiii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D121S;
xiv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167 A and D134S; or
xv. one or more of E84D, E85K and E92Q;
xvi. one or more of E84Q, E85K, E92Q, E97S, D126G and E135S;
xvii. one or more of E85K, E92Q, E94S, E97S and D126G;
xviii. one or more of E76S, E85K, E92Q, E97S and D126G;
xix. one or more of E71S, E85K, E92Q, E97S and D126G;
xx. one or more of D68S, E85K, E92Q, E97S and D126G;
xxi. one or more of E85K, E92Q, E97S and D126G;
xxii. one or more of E84Q, E85K, E92Q, E97S, H103S and D126G;
xxiii. one or more of E84Q, E85K, M90S, E92Q, E97S and D126G;
xxiv. one or more of E84Q, Q87S, E85K, E92Q, E97S and D126G;
xxv. one or more of E84Q, E85S, E92Q, E97S and D126G;
xxvi. one or more of E84S, E85K, E92Q, E97S and D126G;
xxvii. one or more of H81S, E84Q, E85K, E92Q, E97S and D126G;
xxviii. one or more of Y79S, E84Q, E85K, E92Q, E97S and D126G;
xxix. one or more of F70S, E84Q, E85K, E92Q, E97S and D126G;
xxx. one or more of H58S, E84Q, E85K, E92Q, E97S and D126G;
xxxi. one or more of R52S, E84Q, E85K, E92Q, E97S and D126G;
xxxii. one or more of N48S, E84Q, E85K, E92Q, E97S and D126G;
xxxiii. one or more of N46S, E84Q, E85K, E92Q, E97S and D126G;
xxxiv. one or more of M44S, E84Q, E85K, E92Q, E97S and D126G;
xxxv. one or more of E92Q and E97S;
xxxvi. one or more of E84Q, E85K, E92Q and E97S;
xxxvii. one or more of E84Q and E85K;
xxxviii. one or more of E84Q, E85K and D126G;
xxxix. one or more of E84Q, E85K, D126G and E167A;
xl. one or more of E92Q, E97S and D126G;
xli. one or more of E84Q, E85K, E92Q, E97S and D126G;
xlii. one or more of E84Q, E85K, E92Q, E97S and E167A;
xliii. one or more of E84Q, E85K, E92Q, D126G and E167A;
xliv. one or more of E84Q, E85K, E97S, D126G and E167A;
xlv. one or more of E84Q, E92Q, E97S, D126G and E167A;
xlvi. one or more of E85K, E92Q, E97S, D126G and E167A;
xlvii. one or more of E84D, E85K and E92Q;
xlviii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D121S;
xlix. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D68S;
l. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E135S;
li. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E128S;
lii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E102S;
liii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E94S;
liv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E71S;
lv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E50S;
lvi. one or more of E76S, E84Q, E85K, E

Assignees

• Oxford Nanopore Tech Plc

Inventors

• Bruce Mark
• Clarke James Anthony
• Heron Andrew John
• Jayasinghe Lakmal
• Wallace Elizabeth Jayne

Classifications

• C07K14/43536 Primary

  from worms · CPC title

• G01N27/447

  using electrophoresis · CPC title

• C07K2319/55

  containing a fusion with a toxin, e.g. diphteria toxin · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title