Affinity reagents having enhanced binding and detection characteristics
An affinity reagent, having: (a) a retaining component such as a structured nucleic acid particle; and (b) one or both of (i) one or more label components attached to the retaining component, and (ii) one or more binding components attached to the retaining component.
1. A method of detecting an analyte, comprising:
(a) contacting an array of analytes with a detectable probe, wherein the array of analytes comprises individual analytes each attached to sites that are physically isolated from all other analytes in the array of analytes,
wherein the detectable probe comprises:
(i) a retaining component comprising nucleic acid origami,
(ii) two or more label components attached to the retaining component, and
(iii) two or more binding components attached to the retaining component, wherein the two or more binding components recognize the same epitope of the analyte,
wherein the detectable probe binds to the analyte via non-covalent binding of at least one of the two or more binding components to the epitope; and
(b) acquiring signals from the two or more label components of the detectable probe bound to the analyte, thereby detecting the analyte.
2. The method of claim 1 , wherein the nucleic acid origami comprises a scaffold nucleic acid and a staple nucleic acid hybridized to two regions of the scaffold nucleic acid, wherein the two regions of the scaffold nucleic acid are separated by an intervening region that does not hybridize to the staple nucleic acid.
3. The method of claim 2 , wherein the scaffold nucleic acid comprises a circular nucleic acid.
4. The method of claim 2 , wherein the nucleic acid origami comprises a plurality of staple nucleic acids hybridized to the scaffold nucleic acid.
5. The method of claim 1 , wherein the two or more label components comprise fluorophores that emit fluorescence at the same wavelength, and wherein the signals comprise the fluorescence.
6. The method of claim 5 , wherein the retaining component maintains the at least two label components at a spacing of at least 15 nm.
7. The method of claim 1 , wherein the analyte is attached to the site via a structured nucleic acid particle.
8. The method of claim 1 , wherein the analyte comprises a polypeptide.
9. The method of claim 8 , wherein the epitope is a trimer amino acid sequence.
10. The method of claim 8 , wherein the epitope is a tetramer amino acid sequence.
11. The method of claim 8 , wherein the polypeptide is denatured.
12. The method of claim 1 , wherein the two or more binding components comprise antibodies or functional fragments thereof.
13. The method of claim 1 , wherein the two or more binding components comprise nucleic acid aptamers.
14. The method of claim 1 , wherein the structures of the two or more binding components are the same.
15. The method of claim 1 , wherein the sites are separated by at least 100 nm.
16. The method of claim 1 , wherein the detectable probe is bound to the analyte via non-covalent binding of only one binding component of the two or more binding components to the epitope.
17. The method of claim 1 , wherein the retaining component comprises a first nucleic acid sequence that is complementary to a nucleic acid that is coupled to one of the binding components.
18. The method of claim 17 , wherein the retaining component further comprises a second nucleic acid sequence that is complementary to a nucleic acid that is coupled to one of the label components, wherein the first nucleic acid sequence differs from the second nucleic acid sequence.
19. The method of claim 1 , wherein the array of analytes comprises at least 1×10 4 different analytes each attached to sites that are physically isolated from all other analytes in the array of analytes.
20. The method of claim 1 , wherein the structures of the two or more label components are the same.
21. The method of claim 1 , further comprising:
(c) contacting the array of analytes with a second detectable probe,
wherein the second detectable probe comprises:
(i) a second retaining component comprising nucleic acid origami,
(ii) a second label component attached to the second retaining component, and
(iii) a second binding component attached to the second retaining component, wherein the second binding component of the second detectable probe is different from the two or more binding components of the detectable probe,
wherein the second detectable probe binds to the analyte via non-covalent binding of the second binding component to an epitope of the analyte; and
(d) acquiring signals from the second label component of the second detectable probe bound to the analyte, thereby detecting the analyte.
22. The method of claim 21 , further comprising, prior to step (c), removing the detectable probe of step (a) from the analyte.
23. The method of claim 22 , wherein the analyte comprises a polypeptide and wherein the method further comprises (e) identifying the polypeptide based on the detecting of steps (b) and (d).