SCAFFOLD-BASED POLYMERASE ENZYME SUBSTRATES

• US 20120077189A1
• Filed: 08/25/2011
• Published: 03/29/2012
• 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, Sebo, Lubomir, Yue, Stephen, Brogley, Louis, Fedorov, Andrei, Sun, Wei-Chuan
• Granted Patent: US 8,906,612 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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 of the structure:

(DL_D)_n-X-(L_NPN)_m

wherein each D is a fluorescent dye moiety, X is a multifunctional core with multiple linking sites, P is a polyphosphate moiety having 2 to 10 phosphates, N is a nucleoside moiety, m+n=3 or greater, and L_D and L_N are either direct bonds or linkers.

2. The composition of claim 1 comprising a compound of the structure:

3. The composition of claim 1 comprising a compound of the structure:

4. The composition of claim 2 wherein D₁ is a FRET donor and D₂ is a FRET acceptor.

5. The composition of claim 1 comprising a compound of the structure:

6. The composition of claim 5 wherein D₁ and D2 are FRET donors and D₃ is a FRET acceptor.

7. The composition of claim 1 comprising a compound of the structure

8. The composition of claim 1 wherein D₁, D₂, and D₃ are FRET donors and D₄ is a FRET acceptor.

9. The composition of claim 2 wherein X comprises a trifunctional six-membered aromatic moiety.

10. The composition of claim 9 wherein the trifunctional six-membered aromatic moiety comprise either a tri-substituted benzene or a tri-substituted triazine.

22. The composition of claim 1 wherein X comprises the structure:

23. The composition of claim 1 wherein X comprises the structure:

24. The composition of claim 1 wherein X comprises the structure:

28. 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.

29. 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.

11-21. -21. (canceled)

25-27. -27. (canceled)

30. (canceled)

32. The composition of claim 30 wherein m is from 2 to about 10.

31. A composition comprising a compound having the structure:

Y-(L-D-P—

N)_m

wherein Y is a multifunctional core having m linking sites, L is either a direct bond or a linker, D is a fluorescent dye moiety, P is a polyphosphate having from 2 to 10 phosphates, and N is a nucleoside moiety, and m is greater than 2.

33-37. -37. (canceled)

38. 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 either a direct bond or a linker, P is a polyphosphate moiety having 2 to 10 phosphates, N is a nucleoside moiety, and m is from 2 to 8.

39. The composition of claim 38 wherein D comprises a cyanine dye.

40-43. -43. (canceled)

44. A composition comprising a compound having the structure:

45. The composition of claim 44 comprising a compound with the structure:

46. The composition of claim 45 wherein m, n, s, and t=1.

47. The composition of claim 44 wherein A comprises a cyanine dye.

48. The composition of claim 47 wherein each D comprises a cyanine dye.

49-57. -57. (canceled)