Mutant lysenin pores

• US 9,777,049 B2
• Filed: 03/15/2013
• Issued: 10/03/2017
• Est. Priority Date: 04/10/2012
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Bruce, Mark, Clarke, James, Heron, Andrew, Jayasinghe, Lakmal, Wallace, Elizabeth Jayne
• Primary Examiner(s): TSAY, MARSHA M
• Application Number:
  US14/391,660
• Publication Number:
  US 20150068904A1
• Time in Patent Office: 1,663 Days
• Field of Search: None
• CPC Class Codes: C07K 14/43536 from worms
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 27/447 using electrophoresis

Abstract

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

Claims

1. A polypeptide comprising a lysenin monomer comprising a variant of the amino acid sequence of SEQ ID NO:

2, wherein the variant has at least 90% amino acid identity to the amino acid sequence of SEQ ID NO;

2 over a stretch of amino acids from position 44 to position 126 of SEQ ID NO;

2, and wherein the polypeptide comprises amino acid substitutions at positions E84 and E92 of SEQ ID NO;

2, wherein the amino acid substitutions at positions E84 and E92 alter the ability of the polypeptide to interact with a polynucleotide by increasing the net positive charge or neutralizing the negative charges at positions E84 and E92.

2. The polypeptide according to claim 1, wherein the polypeptide further comprises substitutions at positions E85, E97 and D126 of SEQ ID NO:

2 which alter the ability of the monomer to interact with a polynucleotide.

3. The polypeptide according to claim 1, wherein the amino acid(s) substituted at positions E84 and E92 are selected from asparagine (N), serine (S), glutamine (Q), arginine (R), glycine (G), tyrosine (Y), leucine (L), lysine (K) or alanine (A).

4. The polypeptide according to claim 1, wherein the polypeptide comprises amino acid substitutions selected from the group consisting of:

i. one or more of E84Q, E85K, E92Q, E97S, D126G and E167A;

ii. one or more of E76S, E84Q, E85K, E92Q, E97S, D126G and E167A;

iii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E50S;

iv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E71S;

v. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E94S;

vi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E102S;

vii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E128S;

viii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E135S;

ix. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D68S;

x. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D121S;

xi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D134S;

xii. one or more of E84Q, E85K, E92Q, E97S, D126G and E135S;

xiii. one or more of E84Q, E85K, E92Q, E97S, H103S and D126G;

xiv. one or more of E84Q, E85K, M90S, E92Q, E97S and D126G;

xv. one or more of E84Q, Q87S, E85K, E92Q, E97S and D126G;

xvi. one or more of E84Q, E85S, E92Q, E97S and D126G;

xvii. one or more of E84S, E85K, E92Q, E97S and D126G;

xviii. one or more of H81S, E84Q, E85K, E92Q, E97S and D126G;

xix. one or more of Y79S, E84Q, E85K, E92Q, E97S and D126G;

xx. one or more of F70S, E84Q, E85K, E92Q, E97S and D126G;

xxi. one or more of H58S, E84Q, E85K, E92Q, E97S and D126G;

xxii. one or more of R52S, E84Q, E85K, E92Q, E97S and D126G;

xxiii. one or more of N48S, E84Q, E85K, E92Q, E97S and D126G;

xxiv. one or more of N46S, E84Q, E85K, E92Q, E97S and D126G;

xxv. one or more of M44S, E84Q, E85K, E92Q, E97S and D126G;

xxvi. one or more of E84Q, E85K, E92Q and E97S;

xxvii. one or more of E84Q, E85K, E92Q, E97S and D126G;

xxviii. one or more of E84Q, E85K, E92Q, E97S and E167A;

xxix. one or more of E84Q, E85K, E92Q, D126G and E167A;

xxx. one or more of E84Q, E92Q, E97S, D126G and E167A;

xxxi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D121S;

xxxii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D68S;

xxxiii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E135S;

xxxiv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E128S;

xxxv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E102S;

xxxvi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E94S;

xxxvii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E71S;

xxxviii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E50S;

xxxix. one or more of E76S, E84Q, E85K, E92Q, E97S, D126G and E167A;

or

xl. one or more of E84Q, E85K, E92Q, E97S, D126G and E167A.

5. The polypeptide according to claim 4, wherein the polypeptide comprises all of the substitutions in any one of i to xl.

6. The polypeptide according to claim 1, wherein the polypeptide is chemically modified.

7. The polypeptide according to claim 6, wherein

the polypeptide is chemically modified by attachment of a molecule to one or more cysteines, attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus, optionally wherein:

(i) the one or more cysteines or one or more non-natural amino acids have been introduced to the polypeptide by substitution;

(ii) the molecule is (a) a molecular adaptor that facilitates the interaction between a pore comprising the monomer and a target analyte, target nucleotide or target polynucleotide or (b) a polynucleotide binding protein;

(iii) the attachment is via a linker; and

/or

(iv) the molecule is attached to one or more of the positions in the polypeptide that correspond to from about positions 1 to about position 43 and from about position 127 to about position 297 of SEQ ID NO;

2.

8. A construct comprising two or more covalently attached lysenin monomers, where at least one of the monomers is the polypeptide as defined in claim 1.

9. The construct according to claim 8, wherein (i) the two or more monomers are the same or different;

and/or (ii) at least one monomer comprises the sequence shown in SEQ ID NO;

2; and

/or (iii) the construct comprises two monomers; and

/or (iv) the monomers are genetically fused; and

/or (v) the monomers are attached via a linker.

10. A polynucleotide which encodes the construct according to claim 8 or the polypeptide as defined in claim 1.

11. A homo-oligomeric lysenin pore comprising two or more polypeptides according to claim 1.

12. A hetero-oligomeric lysenin pore comprising at least one polypeptide according to claim 1.

13. The hetero-oligomeric pore according to claim 12, wherein (i) the pore comprises at least one monomer comprising the sequence shown in SEQ ID NO:

2;

(ii) the pore comprises (a) one polypeptide as defined in claim 1 and (b) a sufficient number of identical monomers to form the pore, wherein the polypeptide in (a) is different from the identical monomers in (b);

or (iii) the pore comprises only one polypeptide as defined in claim 1.

14. The pore according to claim 11, wherein at least one of the polypeptides is chemically-modified.

15. A pore comprising at least one construct according to claim 8.

16. The pore according to claim 15, wherein at least one of the constructs is chemically-modified.

17. A method of characterising a target analyte, comprising:

(a) contacting the target analyte with a lysenin pore comprising at least one polypeptide according to claim 1 such that the target analyte moves through the pore; and

(b) taking one or more measurements as the analyte moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the target analyte and thereby characterising the target analyte.

18. The method according to claim 17, wherein the target analyte is a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a dye, a bleach, a pharmaceutical, a diagnostic agent, a recreational drug, an explosive or an environmental pollutant.

19. The method according to claim 18, wherein step (a) comprises contacting the target polynucleotide with the pore and a polynucleotide binding protein and the polynucleotide binding protein controls the movement of the target polynucleotide through the pore.

20. The method according to claim 18, wherein characterising the target polynucleotide comprises estimating the sequence of or sequencing the target polynucleotide.

21. A kit for characterizing a target polynucleotide comprising (a) a lysenin pore comprising at least one polypeptide according to claim 1 and (b) a polynucleotide binding protein.

22. An apparatus for characterizing target polynucleotides in a sample, comprising (a) a plurality of lysenin pores, each comprising at least one polypeptide according to claim 1 and (b) a plurality of polynucleotide binding proteins.

23. The apparatus according to claim 22, wherein the apparatus comprises:

(i) a sensor device that is capable of supporting the plurality of lysenin pores and being operable to perform polynucleotide characterisation using the pores and polynucleotide binding 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.

24. A polypeptide comprising a variant of the amino acid sequence of SEQ ID NO:

2, wherein the variant has amino acid substitutions at positions E84 and E92 of SEQ ID NO;

2 and up to 10 modifications (including the E84 and E92 substitutions) within the amino acid sequence from position 44 to position 126 of SEQ ID NO;

2 at positions selected from the group consisting of;

M44, N46, N48, E50, R52, H58, D68, F70, E71, S74, E76, S78, Y79, S80, H81, S82, E85, S86, Q87, S89, M90, E94, E97, E102, H103, T104, T106, R115, Q117, N119, D121, and D126, and wherein the modifications (including the E84 and E92 substitutions) alter the ability of the polypeptide to interact with a polynucleotide by increasing net positive charge or neutralizing negative charges of amino acids at the selected positions.