High intensity labeled reactant compositions and methods for sequencing
Compositions useful for the detection of single molecules in a sample are provided. In some aspects, the disclosure provides a nucleotide connected to a nucleic acid comprising a FRET label comprising at least three luminescent molecules. In some embodiments, the nucleic acids described herein comprise one or more structural features that provide enhanced fluorescence intensity. In some aspects, methods of sequencing using the labeled nucleotides of the disclosure are provided.
1. A labeled nucleotide comprising a nucleoside polyphosphate connected to a nucleic acid comprising a FRET label that comprises at least three luminescent molecules, including at least one donor molecule and at least one acceptor molecule, wherein the FRET label comprises a donor molecule to acceptor molecule ratio of greater than 1:1 or an acceptor molecule to donor molecule ratio of greater than 1:1, and wherein each luminescent molecule is attached at an attachment site that is at least 2 bases separated from a guanine or a cytosine on the nucleic acid.
2. The labeled nucleotide of claim 1 , wherein the FRET label comprises a donor molecule to acceptor molecule ratio of greater than 1.5:1, greater than 2:1, 2.5:1, greater than 3:1, greater than 3.5:1, or greater than 4:1.
3. The labeled nucleotide of claim 1 , wherein the FRET label comprises an acceptor molecule to donor molecule ratio of greater than 1.5:1, greater than 2:1, greater than 2.5:1, greater than 3:1, greater than 3.5:1, or greater than 4:1.
4. The labeled nucleotide of claim 1 , wherein the FRET label comprises at least two donor molecules and at least one acceptor molecule.
5. The labeled nucleotide of claim 1 , wherein the FRET label comprises at least one donor molecule and at least two acceptor molecules.
6. The labeled nucleotide of claim 1 , wherein the nucleic acid comprises one or more stem-loop structures.
7. The labeled nucleotide of claim 6 , wherein at least one donor molecule or at least one acceptor molecule is located in a loop of a stem-loop structure.
8. The labeled nucleotide of claim 6 , wherein the loop region of each stem-loop comprises at least 4 unpaired bases and/or wherein the loop region of each stem-loop comprises a sequence having less than 33% G/C content.
9. The labeled nucleotide of claim 1 , wherein each donor molecule and acceptor molecule are separated by about 1-3 nm.
10. The labeled nucleotide of claim 1 , wherein each donor molecule and acceptor molecule are separated by 3-13 bases.
11. The labeled nucleotide of claim 1 , wherein each donor molecule and acceptor molecule have a FRET efficiency of at least 85%.
12. The labeled nucleotide of claim 1 , wherein the nucleic acid comprises a first oligonucleotide strand and a second oligonucleotide strand hybridized to the first oligonucleotide strand, optionally wherein at least one luminescent molecule is on a different strand from the other luminescent molecules.
13. The labeled nucleotide of claim 1 , wherein one or more luminescent molecules are integrated into the nucleic acid.
14. The labeled nucleotide of claim 1 , wherein the nucleic acid comprises fewer than 150, fewer than 100, or fewer than 50 bases.
15. The labeled nucleotide of claim 1 , wherein one or more luminescent molecules are attached to the nucleic acid at an attachment site that is at least 5 bases separated from any other attachment site.
16. The labeled nucleotide of claim 1 , wherein the nucleic acid comprises DNA, RNA, PNA, LNA, or a derivative thereof.
17. The labeled nucleotide of claim 1 , wherein each luminescent molecule is attached at an attachment site that is at least 3 bases separated from the guanine or the cytosine on the nucleic acid.
18. The labeled nucleotide of claim 1 , wherein at least one luminescent molecule is attached at an abasic site on the nucleic acid.
19. The labeled nucleotide of claim 1 , wherein at least one luminescent molecule is a fluorescent dye, optionally wherein the fluorescent dye is a rhodamine dye, a BODIPY dye, or a cyanine dye.
20. The labeled nucleotide of claim 1 , wherein the nucleotide is separated from any of the luminescent molecules by between approximately 1 and 10 nm.
21. A method of determining the sequence of a template nucleic acid, the method comprising:
(i) exposing a complex in a target volume, the complex comprising the template nucleic acid, a primer, and a polymerizing enzyme, to one or more different types of luminescently labeled nucleotides, wherein each type of luminescently labeled nucleotide comprises a labeled nucleotide according to claim 1 ;
(ii) directing a series of pulses of one or more excitation energies towards a vicinity of the target volume;
(iii) detecting a plurality of emitted photons from luminescently labeled nucleotides during sequential incorporation into a nucleic acid comprising the primer; and
(iv) identifying the sequence of incorporated nucleotides by determining at least one of luminescent intensity and luminescent lifetime based on the emitted photons.
22. A kit for sequencing a template nucleic acid, the kit comprising:
two or more different types of luminescently labeled nucleotides, wherein each type of luminescently labeled nucleotide comprises a labeled nucleotide according to claim 1 .
23. A nucleic acid sequencing reaction composition comprising two or more different types of luminescently labeled nucleotides in a reaction mixture, wherein each type of luminescently labeled nucleotide comprises a labeled nucleotide according to claim 1 .
24. A labeled nucleotide comprising a nucleoside polyphosphate connected to a nucleic acid comprising a FRET label that comprises at least three luminescent molecules, including at least one donor molecule and at least one acceptor molecule, wherein the FRET label comprises a donor molecule to acceptor molecule ratio of greater than 1:1 or an acceptor molecule to donor molecule ratio of greater than 1:1, wherein each luminescent molecule is attached to the nucleic acid at an attachment site on the nucleic acid, and wherein at least one luminescent molecule is attached at an abasic site on the nucleic acid.
25. A method of determining the sequence of a template nucleic acid, the method comprising:
(i) exposing a complex in a target volume, the complex comprising the template nucleic acid, a primer, and a polymerizing enzyme, to one or more different types of luminescently labeled nucleotides, wherein each type of luminescently labeled nucleotide comprises a labeled nucleotide according to claim 24 ;
(ii) directing a series of pulses of one or more excitation energies towards a vicinity of the target volume;
(iii) detecting a plurality of emitted photons from luminescently labeled nucleotides during sequential incorporation into a nucleic acid comprising the primer; and
(iv) identifying the sequence of incorporated nucleotides by determining at least one of luminescent intensity and luminescent lifetime based on the emitted photons.
26. A kit for sequencing a template nucleic acid, the kit comprising:
two or more different types of luminescently labeled nucleotides, wherein each type of luminescently labeled nucleotide comprises a labeled nucleotide according to claim 24 .
27. A nucleic acid sequencing reaction composition comprising two or more different types of luminescently labeled nucleotides in a reaction mixture, wherein each type of luminescently labeled nucleotide comprises a labeled nucleotide according to claim 24 .