Signal amplification of fluorescence in situ hybridization

1. A method of detecting a target nucleic acid, the method comprising:

(a) obtaining a labeled nucleic acid probe for a target nucleic acid, wherein the probe is double-stranded and comprises a capture tag in only one strand of the probe, and wherein the one strand comprising the capture tag is complementary to the target nucleic acid;

(b) contacting both strands of the probe, in denatured form, with the target nucleic acid under conditions suitable for nucleic acid hybridization, to produce a duplex comprising the capture tag and a target nucleic acid;

(c) linking the probe in the duplex to a signal amplification conjugate via the capture tag; and

(d) producing an amplified detectable signal from the signal amplification conjugate in the duplex.

2. The method of claim 1 , wherein the capture tag is an affinity tag.

3. The method of claim 2 , wherein the affinity tag is selected from a hapten and a biotin moiety.

4. The method of claim 1 , wherein the capture tag comprises a chemoselective functional group.

5. The method of claim 4 , wherein the chemoselective functional group is selected from the group consisting of an alkyne, a cyclooctyne, an azide, a phosphine, a maleimide, a thiol, an alkoxyamine and an aldehyde.

6. The method of claim 1 , wherein the labeled nucleic acid probe comprises a PNA, a RNA or a DNA sequence complementary to the target nucleic acid.

7. The method of claim 1 , wherein the labeled nucleic acid probe is at least 20 nucleotides in length.

8. The method of claim 1 , wherein the target nucleic acid is in a cell.

9. The method of claim 8 , wherein the cell is in a tissue section.

10. The method of claim 8 , wherein the cell is in a formalin-fixed, paraffin-embedded (FFPE) tissue section.

11. The method of claim 1 , wherein:

linking step (c) comprises linking the probe in the duplex to an enzyme conjugate via the capture tag to produce an enzyme-labeled duplex; and

producing step (d) comprises incubating the enzyme-labeled duplex with an enzyme substrate, wherein the enzymatic activity of the enzyme conjugate produces the amplified detectable signal.

12. The method of claim 11 , wherein the enzyme substrate comprises a fluorescent compound described by the formula:

(R 1 ) n —(X) q —(R 2 ) m

wherein each R 1 is independently, a phenol-containing group, a ferulic acid, a cinnamic acid or a cinnapinic acid group;

each R 2 is a fluorophore;

each X is a linker group or a chemical bond; and

m, n and q are each independently an integer from 1 to 10.

13. The method of claim 12 , wherein the fluorescent compound is Fer-4-Flu.

14. The method of claim 11 , wherein the enzyme substrate comprises a fluorescent compound and a crosslinker capable of peroxidase-catalyzed radical chain polymerization to produce an insoluble adduct.

15. The method of claim 14 , wherein the crosslinker is selected from 3,3′-diamino benzidin (DAB) and alpha-cyano-4-hydroxy-cinnamic acid (α-CHC).

16. The method of claim 11 , wherein the enzyme conjugate is a peroxidase conjugate and the enzyme substrate is a peroxidase substrate.

17. The method of claim 16 , wherein the peroxidase conjugate comprises an HRP polymer.

18. The method of claim 17 , wherein the peroxidase conjugate is an HRP-azide conjugate or an HRP-cyclooctyne conjugate.

19. The method of claim 17 , wherein the peroxidase enzyme is horse radish peroxidase (HRP) or soybean peroxidase (SP).

20. The method of claim 1 , wherein the target nucleic acid is an RNA transcript.