Protected dye-labeled reagents

• US 10,669,299 B2
• Filed: 11/21/2016
• Issued: 06/02/2020
• Est. Priority Date: 11/20/2015
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Sebo, Lubomir, Osuna, Honey, Yue, Stephen, Lapin, Yuri
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/357,965
• Publication Number:
  US 20170145495A1
• Time in Patent Office: 1,289 Days
• Field of Search: 536 231
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• CPC Class Codes: C09B 69/105 containing a methine or polymethine dye
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

Labeled nucleotide analogs comprising at least one avidin protein, at least one dye-labeled compound, and at least one nucleotide compound are provided. The analogs are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The analogs are detectable with high sensitivity at desirable wavelengths. They contain structural components that modulate the interactions of the analogs with DNA polymerase, thus decreasing photodamage and improving the kinetic and other properties of the analogs in sequencing reactions. Also provided are nucleotide and dye-labeled compounds of the subject analogs, as well as intermediates useful in the preparation of the compounds and analogs. Compositions comprising the compounds, methods of synthesis of the intermediates, compounds, and analogs, and mutant DNA polymerases are also provided.

Claims

1. A dye-labeled compound of structural formula (IIIG):

2. The dye-labeled compound of claim 1, wherein each r″

is independently an integer from 0 to 4.

3. The dye-labeled compound of claim 1, wherein s is an integer from 1 to 4.

4. The dye-labeled compound of claim 1 comprising at least two donor dyes.

5. The dye-labeled compound of claim 4, wherein each donor dye is directly coupled to a donor shield element.

6. The dye-labeled compound of claim 1 comprising at least two acceptor dyes.

7. The dye-labeled compound of claim 6, wherein each acceptor dye is directly coupled to an acceptor shield element.

8. The dye-labeled compound of claim 1 comprising at least four donor dyes and at least two acceptor dyes.

9. The dye-labeled compound of claim 1, comprising at least two shield elements.

10. The dye-labeled compound of claim 1, wherein at least one dye compound linker element comprises a shield element or a side chain element.

11. The dye-labeled compound of claim 1, wherein at least one side chain has a molecular weight of at least 300.

12. The dye-labeled compound of claim 1, wherein all of the side chains have a molecular weight of at least 300.

13. The dye-labeled compound of claim 1, wherein at least one side chain comprises a polyethylene glycol.

14. The dye-labeled compound of claim 1, wherein at least one side chain comprises a negatively-charged component.

15. The dye-labeled compound of claim 14, wherein the negatively-charged component comprises a sulfonic acid.

16. The dye-labeled compound of claim 1, wherein at least one side chain comprises a substituted phenyl group.

17. The dye-labeled compound of claim 16, wherein the at least one side chain comprises the structure:

18. The dye-labeled compound of claim 17, wherein each x is independently an integer from 1 to 4.

19. The dye-labeled compound of claim 1, wherein at least one side chain comprises a triazole.

20. The dye-labeled compound of claim 1, wherein at least one side chain comprises one of the structures illustrated in FIG. 24.

21. The dye-labeled compound of claim 1, wherein the at least one shield element comprises the structure:

22. The dye-labeled compound of claim 1, wherein the at least one shield element comprises one of the structures illustrated in FIG. 20

23. The dye-labeled compound of claim 1, wherein t is 1.

24. The dye-labeled compound of claim 1, wherein at least one B″

comprises a biotin moiety.

25. The dye-labeled compound of claim 24, wherein the at least one B″

comprises a bis-biotin moiety.

26. The dye-labeled compound of claim 1, wherein the at least one acceptor dye or the at least one donor dye is a cyanine dye.

27. The dye-labeled compound of claim 1, wherein the compound does not contain a nucleoside.

28. A labeled reagent composition comprising an avidin protein and the dye-labeled compound of claim 1.

29. The dye-labeled compound of claim 1, wherein at least one B″

comprises a reactive functional group.

30. The dye-labeled compound of claim 1, wherein at least one B″

comprises a residue derived from a reactive functional group.

31. The dye-labeled compound of claim 30, wherein the residue derived from a reactive functional group is an amide moiety.

32. The dye-labeled compound of claim 30, wherein the residue derived from a reactive functional group results from a click reaction.