Multiplexed in situ molecular analyses and programmable molecular probes for regulated single amplification
The present invention generally relates to methods for detecting a target in a sample; methods for modulating the reporting intensity of a labeled target in a sample of fixed cells or tissues; methods for detecting the location of at least two targets in a sample; and related compositions.
1. A method for modulating the reporting intensity of a labeled target in a sample of fixed cells or tissues, the method comprising:
complexing a target-recognition agent to the target, wherein the target-recognition agent comprises a first polynucleotide, to form a first polynucleotide-target complex;
reacting the first polynucleotide-target complex via strand displacement with a branched or dendritic dynamic DNA complex to create a dynamic DNA-target complex;
reacting the dynamic DNA-target complex via strand displacement with a first probe comprising at least one dye and a second polynucleotide to form a first labeling complex;
measuring the reporting intensity of the first labeling complex; and
contacting the sample with a second probe comprising at least one dye and a third polynucleotide, wherein the second probe displaces the first probe via strand displacement to form a second labeling complex;
wherein the second probe carries a different number and/or type of dye molecule than the first probe.
2. The method of claim 1 , wherein at least one dye is a fluorescent dye.
3. The method of claim 1 , where at least one probe comprises at least two dye molecules.
4. The method of claim 3 , wherein the at least two dye molecules are the same dye.
5. The method of claim 3 , wherein the at least two dye molecules are different dyes.
6. The method of claim 1 , wherein the first probe and the second probe comprise different dyes.
7. The method of claim 1 , wherein the first labeling complex and the second labeling complex have a 36:1 to 1:36 ratio of dye molecules.
8. The method of claim 1 , wherein the first labeling complex and the second labeling complex have a 9:1 to 1:9 ratio of dye molecules.
9. The method of claim 1 , wherein at least one polynucleotide is at least one selected from a group consisting of DNA and RNA.
10. The method of claim 1 , wherein the target-recognition agent comprises at least one antibody, peptide, aptamer, enzyme, or self-assembling protein.
11. The method of claim 1 , wherein the first polynucleotide comprises a region that binds to the target.
12. The method of claim 1 , further comprising measuring the reporting intensity of the second labeling complex.