Tagged nucleotides useful for nanopore detection

• US 10,669,580 B2
• Filed: 08/23/2017
• Issued: 06/02/2020
• Est. Priority Date: 08/26/2016
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Inventors: Gremyachinskiy, Dmitriy, Crisalli, Peter, Trans, Andrew, Bhat, Ashwini
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/684,726
• Publication Number:
  US 20180057870A1
• Time in Patent Office: 1,014 Days
• Field of Search: 536 41, 536 231, 536 241, 536 266
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2523/31 Electrostatic interactions, e.g. use of cationic polymers in hybridisation reactions
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

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.

Claims

1. 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 a tag covalently attached to the linker, wherein the tag comprises 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, wherein the negatively-charged polymer moiety comprises a covalently linked chain of from 20 to 50 monomer units selected from the monomer unit structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g), (1h), (1i), (1i), (1k), (1l), (1m), and any combinations thereof

2. The compound of claim 1, wherein the compound has a structural formula (II)

3. The compound of claim 2, 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.

4. The compound of claim 1, wherein the compound has a structural formula (III)

5. The compound of claim 4, wherein the compound has a structural formula (IIIa)

6. The compound of claim 5, wherein R=H, n=4, and p=5.

7. The compound of claim 1, wherein the increased ion flow results in a measured current across the nanopore that is greater than O.C. current, optionally at least 5% greater than O.C. current, at least 10% greater than O.C. current, at least 25% greater than O.C. current, or at least 50% greater than O.C. current.

8. The compound of claim 1, wherein the negatively-charged polymer moiety has an overall negative charge of from (−

25) to (−

50), optionally an overall negative charge of from (−

30) to (−

40), or optionally an overall negative charge of from (−

31) to (−

37).

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

10. The compound 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.

11. The compound of claim 1, wherein the negatively-charged polymer moiety is selected from the group consisting of:

-TF-(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), -TF-(SpC4-F4)₂₈- (SEQ ID NO;

     5), -TT-(SpC7-Pra2)₂₈- (SEQ ID NO;

     6), -(SpC2)₈-T₆-(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).

12. A composition comprising a set of compounds, each compound of the set comprising a different tag which results in a different flow of positive ions through a nanopore when the tag enters the nanopore, wherein at least one of the compounds of the set is a compound according to claim 1.