Protected fluorescent reagent compounds

Patent metadata

Field	Value
Publication number	US-11578093-B2
Application number	US-202017006638-A
Country	US
Kind code	B2
Filing date	Aug 28, 2020
Priority date	Aug 5, 2013
Publication date	Feb 14, 2023
Grant date	Feb 14, 2023

Abstract

Protected fluorescent reagent compounds and their methods of synthesis are provided. The compounds 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 compounds contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements.

First claim

What is claimed is:

1. A method of nucleic acid sequence analysis comprising:
providing an array of optically confined regions, each region comprising an immobilized nucleic acid template/polymerase primer complex and a fluorescent compound in fluid contact with the immobilized nucleic acid template/polymerase primer complex;
illuminating the array of optically confined regions with an excitation wavelength; and
detecting fluorescent signals emitted from each region in the array of optically confined regions;
wherein the fluorescent compound is of structural formula (I):
Z—[S′—B′] m   (I); wherein

Z is a multivalent central core element comprising a fluorescent dye element;
each S′ is independently an intermediate chemical group, wherein at least one S′ comprises a shield element;
each B′ is independently a terminal chemical group, wherein at least one B′ comprises a binding element comprising a nucleotide; and
m is an integer from 2 to 24.

2. The method of claim 1 , wherein the shield element decreases photodamage of the fluorescent compound or of a biomolecule associated with the binding element.

3. The method of claim 1 , wherein the shield element decreases contact between the fluorescent dye element and the binding element.

4. The method of claim 1 , wherein the shield element comprises a plurality of side chains.

5. The method of claim 4 , wherein at least one side chain has a molecular weight of at least 300.

6. The method of claim 4 , wherein at least one side chain comprises a dendrimer.

7. The method of claim 4 , wherein at least one side chain comprises a polyethylene glycol.

8. The method of claim 4 , wherein at least one side chain comprises a negatively-charged component.

9. The method of claim 4 , wherein at least one side chain comprises a substituted phenyl group.

10. The method of claim 4 , wherein at least one side chain comprises a triazole.

11. The method of claim 1 , wherein the shield element comprises an inner layer and an outer layer.

12. The method of claim 1 , wherein at least one B′ comprises a binding element comprising a biotin.

13. The method of claim 1 , wherein at least one B′ comprises a binding element comprising a polyphosphate.

14. The method of claim 1 , wherein Z comprises a branching element.

15. The method of claim 1 , wherein m is an integer from 2 to 8.

16. The method of claim 1 , wherein the fluorescent dye element is a cyanine dye.

17. The method of claim 1 , wherein the fluorescent dye element is a multivalent fluorescent dye element.

18. The method of claim 1 , wherein the fluorescent compound is of structural formula (III):

wherein
X is a non-fluorescent multivalent central core element;
at least one D is a fluorescent dye element;
at least one W is a branching element;
n is an integer from 2 to 6;
each p′ is independently an integer from 1 to 4; and
each p″ is independently an integer from 1 to 4.

19. The method of claim 18 , wherein X comprises a polyamine, a substituted cyclohexane, a substituted 1,3,5-triazine, or a substituted benzene.

20. The method of claim 18 , comprising at least one donor fluorophore and at least one acceptor fluorophore.

21. The method of claim 18 , wherein the shield element comprises a plurality of side chains.

22. The method of claim 21 , wherein at least one side chain has a molecular weight of at least 300.

23. The method of claim 21 , wherein at least one side chain comprises a dendrimer.

24. The method of claim 21 , wherein at least one side chain comprises a polyethylene glycol.

25. The method of claim 21 , wherein at least one side chain comprises a negatively-charged component.

26. The method of claim 21 , wherein at least one side chain comprises a substituted phenyl group.

27. The method of claim 21 , wherein at least one side chain comprises a triazole.

28. The method of claim 18 , wherein the shield element comprises an inner layer and an outer layer.

29. The method of claim 18 , wherein at least one B′ comprises a binding element comprising a biotin.

30. The method of claim 18 , wherein at least one B′ comprises a binding element comprising a polyphosphate.

31. The method of claim 18 , wherein n is an integer from 2 to 4.

32. The method of claim 18 , wherein each p′ is independently an integer from 1 to 3.

33. The method of claim 18 , wherein each p″ is independently an integer from 1 to 3.

34. The method of claim 1 , wherein the shield element does not comprise a protein.

35. The method of claim 1 , wherein each region in the array of optically confined regions comprises a plurality of fluorescent compounds in fluid contact with the immobilized nucleic acid template/polymerase primer complex.

36. The method of claim 35 , wherein each region in the array of optically confined regions comprises four different fluorescent compounds, each compound comprising a different nucleotide and emitting a different fluorescent signal.

Assignees

• Pacific Biosciences California Inc

Inventors

• Sebo Lubomir
• Hanes Jeremiah
• Shen Gene
• Brogley Louis
• Yue Stephen
• Zheng Frank
• Lapin Yuri
• Lyle John
• Osuna Honey
• Fedorov Andrei

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C09B23/083

  five >CH- groups · CPC title

• C09B69/105

  containing a methine or polymethine dye · CPC title

• C07F9/65616

  containing the ring system [IMAGE cpc-sch-C07F-1006.gif] having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs · CPC title

• C07F9/65586

  at least one of the hetero rings does not contain nitrogen as ring hetero atom · CPC title