Compositions and methods for analyte detection
The inventions provided herein relate to detection reagents, compositions, methods, and kits comprising the detection reagents for use in detection, identification, and/or quantification of analytes in a sample. Such detection reagents and methods described herein allow multiplexing of many more labeled species in the same procedure than conventional methods, in which multiplexing is limited by the number of available and practically usable colors.
1. A method for identifying an analyte, comprising:
(a) contacting a cell or tissue sample comprising said analyte with a detection reagent comprising (i) a probe targeting said analyte and (ii) a nucleic acid label coupled to said probe, to permit said probe to bind to said analyte, wherein said nucleic acid label comprises a plurality of subsequences;
(b) generating a set of signal signatures in said cell or tissue sample at least in part by
(i) coupling a first decoder probe to a first subsequence of said plurality of subsequences, wherein said first decoder probe comprises a first detectable label,
(ii) detecting a first signal signature from said first detectable label,
(iii) coupling a second decoder probe to a second subsequence of said plurality of subsequences, wherein said second decoder probe comprises a second detectable label, and
(iv) detecting a second signal signature from said second detectable label; and
(c) processing said set of signal signatures to identify said analyte.
2. The method of claim 1 , wherein said analyte is selected from the group consisting of an antigen, a protein, a peptide, a sugar, a glycoprotein, a peptidoglycan, a lipid, a nucleic acid, a virus, and any combination thereof.
3. The method of claim 1 , wherein said cell or tissue sample is immobilized on a solid substrate or support.
4. The method of claim 1 , wherein said probe is selected from the group consisting of a nucleic acid, an antibody, an antigen binding fragment of an antibody, an antibody-like molecule, an enzyme, an antigen, a small molecule, a protein, a peptide, a peptidomimetic, a sugar, a carbohydrate, a lipid, a glycan, a glycoprotein, an aptamer, and any combination thereof.
5. The method of claim 1 , wherein said probe and said nucleic acid label are conjugated together by at least one linker.
6. The method of claim 5 , wherein said at least one linker is a particle.
7. The method of claim 1 , wherein said first subsequence and said second subsequence of said plurality of subsequences are conjugated together by a nucleotidic linker.
8. The method of claim 1 , wherein said probe is a nucleic acid probe and wherein said probe and said nucleic acid label are conjugate together by a nucleotidic linker.
9. The method of claim 8 , wherein said nucleic acid probe and said nucleic acid label are single-stranded.
10. The method of claim 1 , wherein said probe is a nucleic acid probe and wherein said probe and said nucleic acid label are conjugate together by a phosphodiester bond.
11. The method of claim 1 , wherein said probe comprises a plurality of nucleic acid labels, which plurality of nucleic acid labels comprises said nucleic acid label.
12. The method of claim 11 , wherein said plurality of nucleic acid labels comprise identical nucleic acid sequences.
13. The method of claim 1 , wherein said first detectable label or said second detectable label comprises an optical label.
14. The method of claim 13 , wherein said optical label comprises a small molecule dye, a fluorescent molecule, a quantum dot, a chromophore, a chromogenic molecule, a Raman label, or any combination thereof.
15. The method of claim 1 , wherein said analyte is a protein and said probe is an antibody or an antigen-binding fragment of an antibody that binds said protein.
16. The method of claim 1 , wherein said probe is an aptamer that binds said analyte.
17. The method of claim 1 , further comprising, prior to coupling said second decoder probe to said second subsequence, removing said first signal signature.
18. The method of claim 17 , wherein removing said first signal signature comprises washing, heating, photo-bleaching, displacement, cleavage, enzymatic digestion, quenching, chemical degradation, bleaching, oxidation, or any combination thereof.
19. The method of claim 17 , wherein removing said first signal signature comprises removing said first decoder probe.
20. The method of claim 19 , wherein removing said first decoder probe comprises using a buffer comprising a detergent or a denaturant.
21. The method of claim 1 , wherein said plurality of subsequences form an identifier of said probe and wherein (c) comprises comparing said set of signal signatures with said identifier, wherein an agreement between said set of signal signatures and said identifier identifiers said analyte.
22. The method of claim 1 , wherein said analyte is a ribonucleic acid (RNA) molecule and wherein said probe comprises a nucleic acid sequence that hybridizes to said RNA molecule.
23. The method of claim 1 , wherein said first detectable label or said second detectable label comprises a fluorescent label.
24. The method of claim 1 , wherein said first subsequence and said second subsequence are each 3-50 nucleotides in length.
25. The method of claim 24 , wherein said first subsequence and said second subsequence are each 5-30 nucleotides in length.
26. The method of claim 1 , further comprising, prior to (b), binding an additional detection reagent to said analyte, wherein said additional detection reagent comprises (i) an additional probe and (ii) an additional nucleic acid label comprising an additional plurality of subsequences; and wherein generating said set of signal signatures in said cell or tissue sample further comprises
(i) coupling a third decoder prior to a subsequence of said additional plurality of subsequences, wherein said third decoder probe comprises a third detectable label,
(ii) detecting a third signal signature from said third detectable label,
(iii) coupling a fourth decoder probe to an additional subsequence of said additional plurality of subsequences, wherein said fourth decoder probe comprises a fourth detectable label, and
(iv) detecting a fourth signal signature from said fourth detectable label.
27. A method for identifying an analyte, comprising:
(a) contacting a cell or tissue sample with a nucleic acid detection reagent comprising (i) a probe sequence targeting a ribonucleic acid (RNA) molecule and (ii) a label sequence comprising a plurality of subsequences that form an identifier of said probe;
generating a set of signal signatures in said cell or tissue sample at least in part by
(i) coupling a first decoder probe to a first subsequence of said plurality of subsequences, wherein said first decoder probe comprises a first fluorescent label,
(ii) detecting a first signal signature from said first fluorescent label, wherein said first signal signature is associated with said first subsequence
(iii) coupling a second decoder probe to a second subsequence of said plurality of subsequences, wherein said second decoder probe comprises a second fluorescent label, and
(iv) detecting a second signal signature from said second fluorescent label, wherein said second signal signature is associated with said second subsequence; and
(c) comparing said set of signal signatures with said identifier to identify said RNA molecule.
28. The method of claim 27 , wherein said first subsequence and said second subsequence are each 3-50 nucleotides in length.
29. The method of claim 28 , wherein said first subsequence and said second subsequence are each 5-30 nucleotides in length.