Polypeptide tagged nucleotides and use thereof in nucleic acid sequencing by nanopore detection

Patent metadata

Field	Value
Publication number	US-11739378-B2
Application number	US-202117201834-A
Country	US
Kind code	B2
Filing date	Mar 15, 2021
Priority date	Sep 10, 2015
Publication date	Aug 29, 2023
Grant date	Aug 29, 2023

Abstract

The present disclosure relates to compositions and methods based on polypeptide-tagged nucleotide, and the use of such polypeptide-tagged nucleotides in nanopore devices and methods.

First claim

The invention claimed is:

1. A method for determining the sequence of a nucleic acid comprising:
(a) providing a nanopore sequencing composition comprising: a membrane, an electrode on the cis side and the trans side of the membrane, a nanopore with its pore extending through the membrane, an electrolyte solution in contact with both electrodes, an active polymerase situated adjacent to the nanopore, and a primer strand complexed with the polymerase;
(b) contacting the nanopore sequencing composition with (i) a strand of the nucleic acid; and (ii) a set of tagged nucleotides each with a different tag, wherein each different tag causes a different blocking current and/or blocking voltage and/or has a different dwell time when it is situated in the nanopore, and the set comprises at least one compound of structural formula (I):
N-P-L-T   (I)

wherein,
N is a nucleoside;
P is an oligophosphate covalently attached to a 5′-O group of the nucleoside, wherein the oligophosphate consists of 3 to 12 phosphate groups;
L is a linker covalently attached to a terminal phosphate group of the oligophosphate; and
T is a polypeptide tag covalently attached to the linker, wherein the polypeptide has an overall charge and comprises at least one helical structure, wherein the helical structure is an α-helix comprising the sequence motif (EAAA)16-E5; and

(c) detecting the different blocking currents and/or blocking voltages and/or different dwell times of the tags over time and correlating to each of the different tagged nucleotides incorporated by the polymerase which are complimentary to the nucleic acid sequence, and thereby determining the nucleic acid sequence.

2. The method of claim 1 , wherein the compound comprises structural formula (II):

Base is selected from the group consisting of adenosine, cytidine, guanosine, thymidine, and uridine;
R is selected from the group consisting of H and OH;
n is from 1 to 4;
Linker is a linker comprising a covalently bonded chain of 2 to 100 atoms; and
Polypeptide is a polypeptide tag comprising an overall charge and at least one helical structure.

3. The method of claim 1 , wherein the length of the polypeptide tag is at least 16 amino acid residues.

4. The method of claim 1 , wherein the length of the polypeptide tag is at least 20 amino acid residues.

5. The method of claim 1 , wherein the length of the polypeptide tag is at least 25 amino acid residues.

6. The method of claim 1 , wherein the length of the polypeptide tag is at least 30 amino acid residues.

7. The method of claim 1 , wherein the length of the polypeptide tag is at least 40 amino acid residues.

8. The method of claim 1 , wherein the helical structure comprises at least 8 amino acid residues.

9. The method of claim 1 , wherein the helical structure comprises at least 16 amino acid residues.

10. The method of claim 1 , wherein the helical structure comprises at least 20 amino acid residues.

11. The method of claim 1 , wherein the overall charge of the polypeptide tag is negative, optionally wherein the overall charge of the polypeptide tag is between about −10 and −30.

12. The method of claim 1 , wherein the 25% of the amino acid residues located at the end of the polypeptide tag distal from the linker have a net charge absolute value greater than the net charge absolute value of the 25% of the amino acid residues located at the end of the polypeptide tag proximal to the linker.

13. The method of claim 1 , wherein P consists of from 3 to 9 phosphate groups.

14. The method of claim 1 , wherein P consists of from 4 to 6 phosphate groups.

15. The method of claim 1 , wherein P consists of 6 phosphate groups.

16. The method of claim 1 , wherein the linker comprises a chemical group selected from the group consisting of: ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and combinations thereof.

Assignees

• Roche Molecular Systems Inc
• Roche Sequencing Solutions Inc

Inventors

• Bergmann Frank
• Seidel Christoph
• Trans Andrew
• Gremyachinskiy Dmitriy
• Kuchelmeister Hannes
• Hillringhaus Lars

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C07H19/20

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12Q2525/113

  incorporating modified backbone · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title