Tagged multi-nucleotides useful for nucleic acid sequencing

• US 10,655,174 B2
• Filed: 05/24/2017
• Issued: 05/19/2020
• Est. Priority Date: 05/27/2016
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Inventors: Gremyachinskiy, Dmitriy, Taing, Meng, Ayer, Aruna, Crisalli, Peter J.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/604,611
• Publication Number:
  US 20170342485A1
• Time in Patent Office: 1,091 Days
• Field of Search: 536 41, 536 231, 435 61
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/48721 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

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.

Claims

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);

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

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-dT₅-(BHEB)-dT₁₄-C3;

(dC6P)₂-dT₂₀-C3;

(dT6P)₂-dT₄-(N3CE-dT)₃-dT₁₃-C3; and

(dG6P)₂-dT₆-(Tmp)₆-dT₈-C3;

or (dA6P)₂-dT₄-(idSp-dT)₄-dT₈-C3;

(dC6P)₂-dT₂₀-C3;

(dT6P)₂-dT₄-(N3CE-dT)₃-dT₁₃-C3; and

(dG6P)₂-dT₆-(Tmp)₆-dT₈-C3.

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