Labeled nucleotide analogs, reaction mixtures, and methods and systems for sequencing
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.
1. A labeled nucleotide analog comprising:
a first avidin protein having four subunits, each subunit comprising one biotin binding site;
a first nucleotide compound, bound to the first avidin protein, the first nucleotide compound comprising a polyphosphate element, a nucleoside element, an optional multivalent central core element, a terminal coupling element, and a nucleotide linker element; and
a first dye-labeled compound, bound to the first avidin protein;
wherein the first dye-labeled compound is a compound of structural formula (IIIG):
wherein
each L′ is independently a dye compound linker element;
each S is independently a shield element;
each Dye is independently either an acceptor dye or a donor dye;
each B″ is independently a terminal coupling element;
each p is independently 0 or 1; and
each r″ is independently an integer from 0 to 8;
s is an integer from 1 to 6; and
t is 0 or 1;
wherein the compound of structural formula (IIIG) comprises at least one shield element,
at least one acceptor dye, and at least one donor dye; and
wherein the at least one shield element does not comprise a protein.
2. The labeled nucleotide analog of claim 1 , wherein the first dye-labeled compound and the first nucleotide compound are each bound to the first avidin protein through a biotin moiety.
3. The labeled nucleotide analog of claim 1 , further comprising a second avidin protein and a second nucleotide compound, wherein
the second avidin protein is bound to the first dye-labeled compound through a biotin moiety and to the second nucleotide compound through a biotin moiety.
4. The labeled nucleotide analog of claim 1 , wherein the first avidin protein is avidin, streptavidin, tamavidin, traptavidin, xenavidin, bradavidin, AVR2, AVR4, or a homolog thereof.
5. The labeled nucleotide analog of claim 1 , wherein the donor dye and the acceptor dye are fluorescent dyes.
6. The labeled nucleotide analog of claim 1 , wherein the first dye-labeled compound comprises at least two donor dyes.
7. The labeled nucleotide analog of claim 1 , wherein the first dye-labeled compound comprises at least two acceptor dyes.
8. The labeled nucleotide analog of claim 1 , wherein the first dye-labeled compound comprises at least two donor dyes and at least two acceptor dyes.
9. The labeled nucleotide analog of claim 1 , wherein the dye compound linker element comprises a shield element or a side chain element.
10. The labeled nucleotide analog of claim 1 , wherein the shield element of the first dye-labeled compound decreases photodamage of the first dye-labeled compound or of a biomolecule associated with the analog or increases brightness of the analog.
11. The labeled nucleotide analog of claim 1 , wherein the shield element comprises a plurality of side chains.
12. The labeled nucleotide analog of claim 11 , wherein at least one side chain has a molecular weight of at least 300.
13. The labeled nucleotide analog of claim 11 , wherein all of the side chains have a molecular weight of at least 300.
14. The labeled nucleotide analog of claim 11 , wherein at least one side chain comprises a polyethylene glycol.
15. The labeled nucleotide analog of claim 11 , wherein at least one side chain comprises a negatively-charged component.
16. The labeled nucleotide analog of claim 11 , wherein at least one side chain comprises a substituted phenyl group.
17. The labeled nucleotide analog of claim 11 , wherein at least one side chain comprises a triazole.
18. The labeled nucleotide analog of claim 11 , wherein at least one side chain comprises the structure:
19. The labeled nucleotide analog of claim 1 , wherein the shield element comprises the structure:
wherein each y is independently an integer from 1 to 6.
20. The labeled nucleotide analog of claim 1 , wherein the shield element comprises the structure:
21. The labeled nucleotide analog of claim 1 , comprising at least two terminal coupling elements.
22. The labeled nucleotide analog of claim 1 , wherein the terminal coupling element comprises a biotin moiety.
23. The labeled nucleotide analog of claim 1 , wherein the acceptor dye or dyes or the donor dye or dyes is a cyanine dye.
24. The labeled nucleotide analog of claim 1 , wherein the first dye-labeled compound does not contain a nucleoside.
25. The labeled nucleotide analog of claim 1 , wherein each r″ is independently an integer from 0 to 4.
26. The labeled nucleotide analog of claim 1 comprising at least two donor dyes.
27. The labeled nucleotide analog of claim 1 comprising at least two acceptor dyes.
28. The labeled nucleotide analog of claim 1 comprising at least two donor dyes and at least two acceptor dyes.
29. The labeled nucleotide analog of claim 1 , wherein at least one dye compound linker element comprises a shield element or a side chain element.
30. The labeled nucleotide analog of claim 1 , wherein the at least one shield element comprises a plurality of side chains.
31. The labeled nucleotide analog of claim 30 , wherein at least one side chain has a molecular weight of at least 300.
32. The labeled nucleotide analog of claim 30 , wherein all of the side chains have a molecular weight of at least 300.
33. The labeled nucleotide analog of claim 30 , wherein at least one side chain comprises a polyethylene glycol.
34. The labeled nucleotide analog of claim 30 , wherein at least one side chain comprises a negatively-charged component.
35. The labeled nucleotide analog of claim 30 , wherein at least one side chain comprises a substituted phenyl group.
36. The labeled nucleotide analog of claim 30 , wherein at least one side chain comprises a triazole.
37. The labeled nucleotide analog of claim 30 , wherein at least one side chain comprises the structure:
38. The labeled nucleotide analog of claim 1 , wherein the at least one shield element comprises the structure:
wherein each y is independently an integer from 1 to 6.
39. The labeled nucleotide analog of claim 1 , wherein the at least one shield element comprises the structure:
40. The labeled nucleotide analog of claim 1 , comprising two terminal coupling elements.
41. The labeled nucleotide analog of claim 1 , wherein the terminal coupling element comprises a biotin moiety.
42. The labeled nucleotide analog of claim 1 , wherein the at least one acceptor dye or the at least one donor dye is a cyanine dye.
43. The labeled nucleotide analog of claim 1 , wherein the first dye-labeled compound does not contain a nucleoside.