Tagged nucleotides useful for nanopore detection

Patent metadata

Field	Value
Publication number	US-11008613-B2
Application number	US-202016825086-A
Country	US
Kind code	B2
Filing date	Mar 20, 2020
Priority date	Aug 26, 2016
Publication date	May 18, 2021
Grant date	May 18, 2021

Abstract

The present disclosure relates to compounds comprising a negatively-charged polymer moiety which is capable of entering a nanopore and upon entering a nanopore in the presence of positive ions results in an increased flow of the positive ions through the nanopore. The present disclosure provides methods of preparing the compounds and for their use as nanopore-detectable tags, in particular, for nanopore-based nucleic acid detection and sequencing.

First claim

What is 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 comprising positive ions 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 compounds each comprising a different nucleoside-5′-oligophosphate moiety covalently linked to a tag, wherein each member of the set of compounds has a different tag which results in a different flow of positive ions through a nanopore when the tag enters the nanopore, and at least one of the different tags comprises a negatively-charged polymer moiety which upon entering a nanopore in the presence of positive ions results in an increased flow of the positive ions through the nanopore, wherein the negatively-charged polymer moiety comprises a covalently linked chain of from 20 to 50 monomer unit structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g), (1h), (1i), (1j), (1k), (1l), (1m), and any combinations thereof

and
(c) detecting the different flows of positive ions resulting from the entry of the different tags in the nanopore over time and correlating to each of the different compounds incorporated by the polymerase which are complementary to the nucleic acid sequence, and thereby determining the nucleic acid sequence.

2. The method of claim 1 , wherein the increased flow of the positive ions results in a measured current across the nanopore that is greater than O.C. current.

3. The method of claim 2 , wherein the increased flow of the positive ions results in a measured current across the nanopore that is at least 5% greater than O.C. current.

4. The method of claim 1 , wherein the negatively-charged polymer moiety has an overall negative charge of from (−25) to (−50).

5. The method of claim 1 , wherein the negatively-charged polymer moiety comprises a covalently linked chain of from 25 to 40 monomer units.

6. The method of claim 1 , wherein the negatively-charged polymer moiety comprises a formula (A)m-(B)n-(C)p-(D)q, wherein A, B, C, and D, are monomer units independently selected from the monomer unit structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g), (1h), (1i), (1j), (1k), (1l), and (1m), and each of m, n, p, and q is from 0 to 40, and m+n+p+q is from 20 to 40.

7. The method of claim 1 , wherein the negatively-charged polymer moiety is selected from the group consisting of: -TT-(SpC2) 28 - (SEQ ID NO: 1), -TT-(SpC3) 28 - (SEQ ID NO: 2), -(SpC3) 31 - (SEQ ID NO: 3), -TT-(dSp) 26 -TT- (SEQ ID NO: 4), -TT-(SpC4-F4) 28 - (SEQ ID NO: 5), -TT-(SpC7-Pra2) 28 - (SEQ ID NO: 6), -(SpC2) 8 -T 6 -(SpC2) 16 - (SEQ ID NO: 12), -(SpC2) 8 -(N3CEdT) 7 -(SpC2) 15 - (SEQ ID NO: 13), -(SpC2) 6 -TT-(BHEB)-T-(SpC2) 20 - (SEQ ID NO: 14, -(SpC2) 9 -T-(BHEB) 2 -T-(SpC2) 17 - (SEQ ID NO: 15), and -(SpC2) 8 -(S500) 3 -(SpC2) 17 - (SEQ ID NO: 16).

8. The method of claim 1 , wherein the at least one compound of the set with a tag comprising a negatively-charged polymer moiety is a 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 the tag covalently attached to the linker.

9. The method of claim 1 , wherein the at least one compound of the set with a tag comprising a negatively-charged polymer moiety is a compound of structural formula (II)

wherein,
Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine;
R is selected from H and OH;
n is from 1 to 4;
Linker is a linker comprising a covalently bonded chain of 2 to 100 atoms; and
Tag is the tag.

10. The method of claim 9 , 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 any combination thereof.

11. The method of claim 1 , wherein the at least one compound of the set with a tag comprising a negatively-charged polymer moiety is a compound of structural formula (III)

wherein,
Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine;
R is selected from H and OH;
n is from 1 to 4;
L B -X-L A is the linker, wherein (a) L A and L B each independently comprises a chemical moiety selected from the group consisting of: linear (C 1 -C 12 ) alkyl, linear (C 1 -C 12 ) alkene, linear (C 1 -C 12 ) alkyne, ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and combinations thereof; and (b) X comprises a chemical moiety selected from the group consisting of ester, ether, thioether, amine, amide, imide, carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime, triazole, and dihydropyridazine; and

Tag is the tag.

12. The method of claim 11 , wherein the compound has a structural formula (IIIa)

wherein,
Base is selected from adenosine, cytidine, guanosine, thymidine, and uridine;
R is selected from H and OH;
n is from 1 to 4;
p is from 2 to 10; and
Tag is the tag comprising the negatively-charged polymer moiety.

13. The method of claim 12 , wherein R═H, n=4, and p=5.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Gremyachinskiy Dmitriy
• Crisalli Peter
• Trans Andrew
• Bhat Ashwini

Classifications

• C07H19/10

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

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6823

  Release of bound markers · CPC title