Modified enzymes

Patent metadata

Field	Value
Publication number	US-11180741-B2
Application number	US-201515517592-A
Country	US
Kind code	B2
Filing date	Oct 6, 2015
Priority date	Oct 7, 2014
Publication date	Nov 23, 2021
Grant date	Nov 23, 2021

Abstract

The invention relates to modified Dda helicases which can be used to control the movement of polynucleotides and are particularly useful for sequencing polynucleotides.

First claim

The invention claimed is:

1. A composition for characterizing a target polynucleotide comprising a transmembrane pore and a DNA-dependent ATPase (Dda helicase in which:
(a) the Dda helicase comprises a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 8 and is recombinantly substituted in at least one residue corresponding to at least one of the following amino acid positions in SEQ ID NO: 8 which interacts with one or more nucleotides in single stranded DNA (ssDNA): H82, N88, P89, F98, D121, V150, P152, F240, F276, S287, H396 and/or Y415; and
(b) the part of the Dda helicase which interacts with the transmembrane pore comprises one or more modifications at one or more residues corresponding to a position in SEQ ID NO: 8 selected from the group consisting of: 3, 4, 5, 176, 177, 179, 180, 185, 193, 194, 195, 198, 199, 200, 202, 203, 204, 207, 208, 209, 210, 211, 212, 213, 216, 221, 224, 255, 318, 347, 405, 415, 434, 437, and 438.

2. The composition according to claim 1 , wherein in (a) the at least one amino acid interacts with the sugar and/or base of the one or more nucleotides in single stranded DNA (ssDNA) is substituted with an amino acid which comprises a larger side chain (R group).

3. The composition according to claim 2 wherein the at least one amino acid which interacts with the sugar and/or base of one or more nucleotides in ssDNA is at the residue corresponding to H82, N88, P89, F98, D121, V150, P152, F240, F276, S287, and H396 in SEQ ID NO: 8.

4. The composition according to claim 2 , wherein the at least one amino acid which interacts with the sugar and/or base of one or more nucleotides in ssDNA is at least one amino acid which intercalates between the nucleotides in ssDNA.

5. The composition according to claim 4 , wherein the at least one amino acid which intercalates between the nucleotides in ssDNA is at a residue corresponding to at least one of P89, F98, and V150 in SEQ ID NO: 8.

6. The composition according to claim 2 , wherein the larger side chain (R group) contains an increased number of carbon atoms, has an increased length, has an increased molecular volume and/or has an increased van der Waals volume.

7. The composition according to claim 2 , wherein the larger side chain (R group) increases the (i) electrostatic interactions, (ii) hydrogen bonding and/or (iii) cation-pi (cation-π) interactions between the at least one amino acid and the one or more nucleotides in ssDNA.

8. The composition according to claim 2 , wherein the amino acid which comprises a larger side chain (R group) is not alanine (A), cysteine (C), glycine (G), selenocysteine (U), methionine (M), aspartic acid (D) or glutamic acid (E).

9. The composition according to claim 1 , wherein
histidine (H) is substituted with arginine (R), lysine (K), glutamine (Q), asparagine (N), phenylalanine (F), tyrosine (Y) or tryptophan (W);
asparagine (N) is substituted with arginine (R), lysine (K), glutamine (Q), histidine (H), phenylalanine (F), tyrosine (Y) or tryptophan (W);
proline (P) is substituted with arginine (R), lysine (K), glutamine (Q), asparagine (N), threonine (T), histidine (H), tyrosine (Y), phenylalanine (F), tryptophan (W), leucine (L), valine (V) or isoleucine (I);
phenylalanine (F) is substituted with arginine (R), lysine (K), histidine (H), tyrosine (Y) or tryptophan (W);
aspartic acid (D) is substituted with arginine (R), lysine (K), glutamine (Q), asparagine (N), histidine (H), phenylalanine (F), tyrosine (Y) or tryptophan (W);
valine (V) is substituted with arginine (R), lysine (K), glutamine (Q), asparagine (N), histidine (H), phenylalanine (F), tyrosine (Y), tryptophan (W), isoleucine (I) or leucine (L);
serine (S) is substituted with arginine (R), lysine (K), glutamine (Q), asparagine (N), histidine (H), phenylalanine (F), tyrosine (Y), tryptophan (W), isoleucine (I) or leucine (L); and/or
tyrosine (Y) is substituted with arginine (R), lysine (K) or tryptophan (W).

10. The composition according to claim 1 , wherein the helicase-sequence comprises a substitution corresponding to:

H82N;

H82Q;

H82W;

N88R;

N88H;

N88W;

N88Y;

P89L;

P89V;

P89I;

P89E;

P89T;

P89F;

D121H;

D121Y;

D121K;

V150I;

V150L;

V150N;

V150W;

V150H;

P152W;

P152F;

P152Y;

P152H;

P152I;

P152L;

P152V;

F240W;

F240Y;

F240H;

F276W;

F276R;

F276K;

F276H;

S287K;

S287R;

S287W;

S287F;

H39

Assignees

• Oxford Nanopore Tech Ltd

Inventors

• Heron Andrew John
• Bowen Rebecca Victoria
• Bruce Mark
• Jayasinghe Lakmal
• Lloyd Joseph Hargreaves
• Soeroes Szabolcs
• Wallace Elizabeth Jayne
• Youd Christopher Peter

Classifications

• C12Y306/04012

  DNA helicase (3.6.4.12) · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• C12N9/14 Primary

  Hydrolases (3) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title