Binding-induced hairpin detection system

A method of detecting a target molecule in a sample, the method comprising:

• a. providing the sample;
• b. incubating the sample with
• i. a first probe comprising a targeting molecule and an oligonucleotide that has a free end comprising a hairpin loop and a hairpin stem and an end attached to the targeting molecule;
• and
• ii. a second probe comprising a targeting molecule and an oligonucleotide that has an end attached to the targeting molecule and a free end comprising a nucleotide sequence that is complementary to a nucleotide sequence at or near the free end of the oligonucleotide of the first probe;
• wherein the targeting molecule of the first and second probe is independently selected from a peptide aptamer, a protein receptor molecule, an antibody, or a ligand that is a peptide, a hormone, a lipid, or a glycan;
• c. contacting the sample with the first probe and the second probe, wherein upon binding of the targeting molecule to a target molecule, the free end of the oligonucleotide of the second probe hybridizes at or near the free end of the oligonucleotide of the first probe forming a hairpin stem, the non-hybridized portions of the first and second probes together with the target molecule bound thereto forming a hairpin loop, thereby providing a binding-induced hairpin with a closed circular structure;
• and
• d. detecting the binding-induced hairpin.

A binding-induced hairpin detection system comprising:

• a. a first probe comprising a targeting molecule and an oligonucleotide that has a free end comprising a hairpin loop and a hairpin stem and an end attached indirectly to the targeting molecule via a linker;
• and
• b. a second probe comprising a targeting molecule and an oligonucleotide that has an end attached indirectly to the targeting molecule via a linker, and a free end comprising a nucleotide sequence that is complementary to a nucleotide sequence at or near the free end of the oligonucleotide of the first probe;
• wherein the targeting molecule of the first and second probe is independently selected from an aptamer, a receptor, an antibody, or a ligand that is a peptide, a hormone, a lipid, or a glycan;
• wherein the linker of the first and second probe is independently selected from a peptide linker or a binding pair, said binding pair comprising a first member attached to the probe and a second member attached to the targeting molecule;
• and
• wherein upon binding of the targeting molecule to a target molecule, the free end of the oligonucleotide of the second probe hybridizes at or near the free end of the oligonucleotide of the first probe forming a hairpin stem, the non-hybridized portions of the first and second probes together with the target molecule bound thereto forming a hairpin loop, thereby providing a binding-induced hairpin with a closed circular structure.