Scaffold-based polymerase enzyme substrates

• US 9,702,001 B2
• Filed: 11/03/2014
• Issued: 07/11/2017
• Est. Priority Date: 08/25/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Shen, Gene, Yue, Stephen, Sun, Wei-Chuan, Fedorov, Andrei, Sebo, Lubomir, Brogley, Louis
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/531,506
• Publication Number:
  US 20150307933A1
• Time in Patent Office: 981 Days
• Field of Search: 536 266, 435 61
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 401/14 containing three or more hetero rings
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07K 5/0815 with the first amino acid being basic
• CPC Class Codes: C07K 5/1019 with the first amino acid being basic
• CPC Class Codes: C09B 23/0025 the substituent being bound through an oxygen atom
• CPC Class Codes: C09B 23/0033 the substituent being bound through a sulfur atom
• CPC Class Codes: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 23/105 two >CH- groups
• CPC Class Codes: C09B 23/164 containing one nitrogen atom
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 2333/9126 DNA-directed DNA polymerase (2.7.7.7)
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The invention provides a novel class of scaffold-based labeled polymerase enzyme substrates. The polymerase enzyme substrates have a multivalent core or scaffold to which is attached fluorescent dye moieties and nucleoside phosphate moities. The polymerase enzyme substrates have multiple fluorescent dye moities and/or multiple nucleoside phosphate moieties. Preferred multivalent cores comprise trifunctional six membered aromatic moities. The invention also provides for sequencing methods and kits with scaffold-based labeled polymerase enzyme substrates.

Claims

1. A composition comprising a compound having the structure:

D-(L-P-N)_m

wherein D is a fluorescent dye moiety having m linking sites, L is a linker, P is a polyphosphate moiety having the structure;

2. The composition of claim 1 wherein D comprises a cyanine dye.

3. The composition of claim 1 wherein m is 3 or 4.

4. The composition of claim 1 wherein the compound has 2 to 40 sulfonate groups.

5. The composition of claim 1 wherein the compound is water soluble.

6. The composition of claim 1 wherein L is a linker having a linear or branched alkyl chain with 2 to 20 carbons.

7. The composition of claim 1 wherein each L-P-N in the compound is the same.

8. The composition of claim 1 wherein each N is A, G, C, T, or U.

9. The composition of claim 1 wherein D comprises a core having multiple covalently connected dyes.

10. The composition of claim 9 wherein number of dyes in the core is from 1 to about 12.

11. The composition of claim 1 wherein linker groups, L, comprise substituted or unsubstituted C₆-C₃₀ alkyl groups, polyols, polyethers, polyamines, amino acids, peptides, saccharides, or combinations thereof.

12. The composition of claim 1 wherein at least one of the linker groups, L, comprises one or more proline, pyrrolidine, or alkynyl moiety.

13. A sequencing mixture comprising a set of 4 nucleotide analog substrates, each having one of the bases A, G, C, T, or A, G, C, U, at least two of the substrates having the structure of claim 1, and each substrate having at least one fluorescent dye moiety different from the fluorescent dye moieties on the other three substrates.

14. The sequencing mixture of claim 13 wherein D comprises a cyanine dye.

15. The sequencing mixture of claim 13 wherein the compound has 2 to 40 sulfonate groups.

16. The sequencing mixture of claim 13 wherein L is a linker having a linear or branched alkyl chain with 2 to 20 carbons.

17. The sequencing mixture of claim 13 wherein each L-P-N in the compound is the same.

18. The sequencing mixture of claim 13 wherein D comprises a core having multiple covalently connected dyes.

19. The sequencing mixture of claim 18 wherein number of dyes in the core is from 1 to about 12.

20. The sequencing mixture of claim 13 wherein linker groups, L, comprise substituted or unsubstituted C₆-C₃₀ alkyl groups, polyols, polyethers, polyamines, amino acids, peptides, saccharides, or combinations thereof.

21. A method for nucleic acid sequencing comprising:

immobilizing a polymerase enzyme complex comprising a polymerase enzyme and a template nucleic acid;

exposing the immobilized polymerase enzyme complex to a plurality of labeled nucleotide analog substrates, at least one of the substrates comprising a compound having the structure of claim 1;

detecting the incorporation of substrates by observing fluorescence from the nucleotide analog substrates; and

using the detected incorporation over time to obtain sequence information about the template nucleic acid.

22. The method of claim 21 wherein D comprises a cyanine dye.

23. The method of claim 21 wherein the compound has 2 to 40 sulfonate groups.

24. The method of claim 21 wherein L is a linker having a linear or branched alkyl chain with 2 to 20 carbons.

25. The method of claim 21 wherein each L-P-N in the compound is the same.

26. The method of claim 21 wherein D comprises a core having multiple covalently connected dyes.

27. The method of claim 26 wherein number of dyes in the core is from 1 to about 12.

28. The method of claim 21 wherein linker groups, L, comprise substituted or unsubstituted C₆-C₃₀ alkyl groups, polyols, polyethers, polyamines, amino acids, peptides, saccharides, or combinations thereof.