Tagged multi-nucleotides useful for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-10655174-B2
Application number	US-201715604611-A
Country	US
Kind code	B2
Filing date	May 24, 2017
Priority date	May 27, 2016
Publication date	May 19, 2020
Grant date	May 19, 2020

Abstract

The present disclosure relates to tagged multi-nucleotide compounds, which comprise a single tag moiety covalently linked to a plurality of nucleoside-5′-oligophosphate moieties. As disclosed herein, these tagged multi-nucleotide compounds have improved characteristics as polymerase substrates and can be used in a range of nucleic acid detection and sequencing methods, including nanopore sequencing-by-synthesis.

First claim

What is claimed is:

1. A compound comprising a single tag covalently linked to a plurality of nucleoside-5′-oligophosphate moieties, wherein the tag is a molecular moiety capable of entering into, becoming positioned in, being captured by, translocating through, and/or traversing a nanopore and producing a nanopore detectable signal, and each nucleoside-5′-oligophosphate moiety is capable of being a substrate for a polymerase, wherein the compound has structural formula (IIIa), (IIIb), or (IIIc):

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 a molecular moiety capable of producing a detectable signal.

2. The compound of claim 1 , wherein the compound has structural formula (IIId), (IIIe), or (IIIf):

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 a molecular moiety capable of producing a detectable signal.

3. The compound of claim 1 , wherein the Tag comprises a molecular moiety selected from the group consisting of a polyethylene-glycol (PEG) oligomer, an organic dye moiety, an oligonucleotide comprising natural and/or non-natural analog monomer units, a polypeptide comprising natural and/or non-natural analog monomer units, and an oligomeric moiety comprising a combination of any of these.

4. The compound of claim 1 , wherein the Tag comprises an oligonucleotide having a structure selected from Table 3, 7, 8, or 10, or a sequence selected from SEQ ID NO: 1-109.

5. The compound of claim 1 , wherein the Tag comprises a polypeptide having a structure selected from Table 5, or a sequence selected from SEQ ID NO: 110-123.

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

7. The compound of claim 1 , wherein the linker comprises a chemical group having structural formula (XVd) or (XVe).

8. A composition comprising a set of compounds according to claim 1 , wherein each compound in the set has a different tag, wherein each different tag causes a different detectable signal.

9. The composition of claim 8 , wherein at least one of the different tags comprises an oligonucleotide.

10. The composition of claim 8 , wherein at least one of the different tags comprises an oligonucleotide having a structure selected from Table 3, 7, 8, or 10, or a sequence selected from SEQ ID NO: 1-109.

11. The composition of claim 8 , wherein the set of compounds comprises (dA6P) 2 -dT 5 -(BHEB)-dT 14 -C3; (dC6P) 2 -dT 20 -C3; (dT6P) 2 -dT 4 -(N3CE-dT) 3 -dT 13 -C3; and (dG6P) 2 -dT 6 -(Tmp) 6 -dT 8 -C3; or (dA6P) 2 -dT 4 -(idSp-dT) 4 -dT 8 -C3; (dC6P) 2 -dT 20 -C3; (dT6P) 2 -dT 4 -(N3CE-dT) 3 -dT 13 -C3; and (dG6P) 2 -dT 6 -(Tmp) 6 -dT 8 -C3.

12. The composition of claim 8 , wherein at least one of the different tags comprises a polypeptide.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Gremyachinskiy Dmitriy
• Taing Meng
• AYER Aruna
• Crisalli Peter J

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• G01N33/48721

  Investigating individual macromolecules, e.g. by translocation through nanopores (Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68) · CPC title

• C07H19/10

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

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title