Methods for multiplex imaging using labeled nucleic acid imaging agents

1. A method for detecting two or more targets, comprising:

(1) contacting a sample being tested for the presence of two or more targets with two or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand and wherein target-specific binding partners of different specificity are linked to different docking strands;

(2) optionally removing unbound target-specific binding partners;

(3) contacting the sample with enzyme-labeled strands capable of binding a docking strand, wherein the enzyme-labeled strands comprise horseradish peroxidase (HRP)-labeled strands;

(4) optionally removing unbound enzyme-labeled strands;

(5) contacting the sample with a substrate for the enzyme comprising a tyramide moiety linked to a fluorophore;

(6) allowing an enzymatic reaction to produce an amplification product;

(7) quenching the enzymatic reaction by removing the bound enzyme-labeled strands;

(8) repeating steps (3)-(7),

(9) after step (8), imaging the sample to detect the presence or absence of two or more targets; and

(10) optionally repeating at least some of steps (1)-(9).

2. The method of claim 1 , wherein the bound enzyme-labeled strands are removed by enzymatically cleaving, modifying, or degrading the enzyme-labeled strands.

3. The method of claim 1 , wherein the bound enzyme-labeled strands are removed by photochemically cleaving the enzyme-labeled strands.

4. The method of claim 1 , further comprising removing or inactivating the amplification product after imaging.

5. The method of claim 1 , wherein the enzyme is capable of converting the tyramide moiety to an activated state capable of binding to tyrosine residues, resulting in deposition of the amplification product on or near the target.

6. The method of claim 1 , wherein the target-specific binding partner is an antibody or a fragment thereof.

7. The method of claim 1 , wherein the docking strand is a nucleic acid strand.

8. The method of claim 7 , wherein the enzyme-labeled strand has a nucleic acid sequence complementary to a corresponding sequence of the docking strand.

9. The method of claim 1 , wherein at least 5 targets are tested for, using a corresponding target-specific binding partner for each target.

10. The method of claim 1 , wherein at least 3 targets are imaged using at least 3 labels in the same imaging step.

11. The method of claim 1 , wherein at least 3 targets are imaged using at least 3 labels, the signal extinguished, and then at least one more target is imaged using at least one of the same labels, wherein the imaging steps may be performed in either order.

12. A method for detecting one or more targets, comprising:

(1) contacting a sample having a target site being tested for the presence of one or more targets with one or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand, and wherein target-specific binding partners of different specificity are linked to different docking strands;

(2) optionally removing unbound target-specific binding partners;

(3) contacting the sample with enzyme-labeled strands having a nucleotide sequence that is complementary to a docking strand, wherein the enzyme-labeled strands comprise horseradish peroxidase (HRP)-labeled strands;

(4) optionally removing unbound enzyme-labeled strands;

(5) contacting the sample with tyramide-bound docking strands, wherein the docking strand is a barcode for an imager strand;

(6) enzymatically converting the tyramide moiety into an activated state, wherein the activated state results in a covalent linkage of the tyramide-bound docking strand to the target site;

(7) removing the enzyme-labeled strands;

(8) optionally repeating a subset of steps 3-7;

(9) contacting the sample with one or more imager strands;

(10) imaging the sample to detect the presence or absence of one or more targets; and

(11) optionally repeating at least some of steps 3-10.

13. The method of claim 12 , wherein the docking strand of the tyramide-bound docking strands comprises a nucleotide sequence complementary to the imager strand.

14. The method of claim 12 , wherein the docking strand linked to the target-specific binding partner introduced in step (1) is different from the docking strand of the tyramide-bound docking strands introduced in step (5).

15. A method for detecting two or more targets, comprising:

(1) contacting a sample being tested for the presence of two or more targets with two or more target-specific binding partners, wherein each target-specific binding partner is linked to a docking strand and wherein target-specific binding partners of different specificity are linked to different docking strands;

(2) optionally removing unbound target-specific binding partners;

(3) contacting the sample with enzyme-labeled strands capable of binding a docking strand wherein the enzyme-labeled strands comprise horseradish peroxidase (HRP)-labeled strands;

(4) optionally removing unbound enzyme-labeled strands;

(5) contacting the sample with a substrate for the enzyme comprising a tyramide moiety linked to a fluorophore;

(6) allowing an enzymatic reaction to produce an amplification product;

(7) quenching the enzymatic reaction by removing the bound enzyme-labeled strands;

(8) imaging the sample to detect the presence or absence of two or more targets; and

(9) optionally repeating at least some of steps (1)-(8).