Binding-induced hairpin detection system

• US 9,176,123 B2
• Filed: 08/30/2010
• Issued: 11/03/2015
• Est. Priority Date: 08/31/2009
• Status: Active Grant

• Current Assignee: The Governors of the University of Alberta (University of Alberta)
• Original Assignee: The Governors of the University of Alberta (University of Alberta)
• Inventors: Le, Xiaochun, Zhang, Hongquan, Li, Xing-Fang
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US13/392,940
• Publication Number:
  US 20120156678A1
• Time in Patent Office: 1,891 Days
• Field of Search: 435/6.1, 435/91.2, 536/22.1
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching

Abstract

This application relates to a binding-induced hairpin detection system comprising: a. a first probe comprising a targeting molecule and an oligonucleotide that has a free end and an end attached to the targeting molecule; and b. 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 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.

Claims

1. 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 to the targeting molecule; and

b. 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; 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.

2. The detection system according to claim 1, wherein nucleotides at or near the free end of the oligonucleotide of the first probe and nucleotides at the free end of the oligonucleotide of the second probe comprise five to eight contiguous complementary nucleotides.

3. The detection system according to claim 1, wherein the targeting molecule is attached to the oligonucleotide via a linker.

4. The detection system according to claim 1, wherein the targeting molecule of the first probe and the targeting molecule of the second probe have distinct target specificities.

5. 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.

6. The method according to claim 5, further comprising incubating the sample with a ligase.

7. The method according to claim 6, wherein the binding-induced hairpin is detected by PCR amplification.

8. The method according to claim 5, further comprising incubating the sample with at least one blocking agent.

9. The method according to claim 5, wherein the targeting molecule is attached to the oligonucleotide via a linker.

10. The method according to claim 5, wherein the targeting molecule of the first probe and the targeting molecule of the second probe bind to the same target molecule or to two separate but associated target molecules.

11. The method according to claim 5, wherein the target molecule is an enzyme, a toxin, a cell receptor, a ligand, a viral or bacterial protein or antigen, a signal transducing agent, a cytokine, an antibody, a growth factor or a disease associated protein or antigen.

12. 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.

13. The detection system according to claim 12, wherein the first or second member of the binding pair is biotin and the other of the first or second member of the binding pair is streptavidin.

14. The detection system according to claim 12, wherein nucleotides at or near the free end of the oligonucleotide of the first probe and nucleotides at the free end of the oligonucleotide of the second probe comprise five to eight contiguous complementary nucleotides.

15. The detection system according to claim 12, wherein the targeting molecule of the first probe and the targeting molecule of the second probe have distinct target specificities.