Hybridization chain reaction
The present invention relates to the use of nucleic acid probes to identify analytes in a sample. In the preferred embodiments, metastable nucleic acid monomers are provided that associate in the presence of an initiator nucleic acid. Upon exposure to the initiator, the monomers self-assemble in a hybridization chain reaction. The initiator nucleic acid may be, for example, a portion of an analyte to be detected or may be part of an initiation trigger such that it is made available in the presence of a target analyte.
1. A method for detecting an analyte in a sample by hybridization chain reaction, the method comprising:
a) contacting the sample with a first nucleic acid hairpin monomer comprising:
i) a first duplex region comprising second and third complementary stem regions, wherein the second complementary region is substantially complementary to a first complementary region of an initiator,
ii) a first hairpin loop region at an end of the first duplex region, and
iii) an initiator complement region that comprises a sticky end and that is substantially complementary to an initiation region of the initiator,
b) contacting the sample with a second nucleic acid hairpin monomer comprising:
i) a second duplex region comprising fourth and fifth complementary stem regions,
ii) a second sticky end region that is substantially complementary to the first hairpin loop region of the first monomer, and
iii) a second hairpin loop region that is substantially complementary to the sticky end of the initiator complement region of the first monomer; and
c) detecting the analyte in the sample by identifying polymers comprising the first and second monomers,
wherein the initiator is able to hybridize with the initiator complement region of the first monomer when analyte is present in the sample and wherein the first and second monomers polymerize upon the hybridization of the initiator with the initiator complement region of the first monomer.
2. The method of claim 1 , wherein the initiator is a nucleic acid.
3. The method of claim 1 , wherein the analyte comprises the initiator.
4. The method of claim 1 , wherein the sample is additionally contacted with an initiation trigger.
5. The method of claim 4 , wherein the initiation trigger comprises an aptamer.
6. The method of claim 5 , wherein the aptamer is able to specifically bind the analyte.
7. The method of claim 5 , wherein the initiation trigger comprises the initiator.
8. The method of claim 7 , wherein the initiator is able to hybridize to the first monomer when the aptamer is bound by the analyte.
9. The method of claim 1 , wherein at least one of the first and second monomers is fluorescently labeled.
10. The method of claim 1 , wherein the analyte is a nucleic acid associated with a pathogen.
11. The method of claim 1 , wherein the sample is obtained from a patient.
12. The method of claim 1 , wherein the first and second nucleic acid hairpin monomers are metastable monomers.
13. The method of claim 1 , wherein the first loop region comprises from about 3 to about 30 nucleotides.
14. The method of claim 1 , wherein the initiator is a single stranded nucleic acid.
15. The method of claim 1 , wherein the polymers are detected by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, mass spectrometry, light scattering spectroscopy, colorimetry and fluorescent spectroscopy.
16. The method of claim 1 , additionally comprising contacting the sample with a third and fourth nucleic acid hairpin monomers wherein:
the third nucleic acid hairpin monomer comprises a sixth complementary region that is complementary to a portion of the first and/or second monomer; and
the fourth nucleic acid monomer comprises a seventh complementary region that is complementary to a portion of the third monomer.
17. The method of claim 16 , wherein the first monomer comprises a bulge region that is complementary to a portion of the third monomer.
18. The method of claim 1 , wherein the second and third complementary regions in the first monomer are identical to the fourth and fifth complementary regions, respectively, of the second monomer.
19. A method for detecting an analyte in a sample, the method comprising:
a) contacting the sample with a first nucleic acid hairpin monomer comprising:
i) a first sticky end, and
ii) a first hairpin loop region;
b) contacting the sample with a second nucleic acid hairpin monomer comprising:
i) a second sticky end region that is substantially complementary to the first hairpin loop region of the first monomer, and
ii) a second hairpin loop region that is substantially complementary to the first sticky end of the first monomer,
wherein the first and second nucleic acid hairpin monomers polymerize via sequential nucleation and opening of alternating first and second hairpins in the presence of the analyte; and
c) detecting the analyte in the sample by identifying polymers comprising the first and second monomers.
20. The method of claim 19 , wherein the analyte comprises an initiator that comprises an initiation region, wherein the initiation region is substantially complementary to the first sticky end of the first monomer.
21. The method of claim 19 , wherein at least one of the first and second monomers is fluorescently labeled.
22. The method of claim 19 , wherein the analyte is a nucleic acid associated with a pathogen.
23. A method for detecting an analyte in a sample by hybridization chain reaction, the method comprising:
contacting the sample with a first nucleic acid hairpin monomer comprising a first hairpin loop region;
contacting the sample with a second nucleic acid hairpin monomer comprising a sticky end region that is substantially complementary to the first hairpin loop region of the first monomer, wherein the first and second monomers polymerize in the presence of the analyte; and
detecting the analyte in the sample by identifying polymers comprising the first and second monomers.
24. The method of claim 23 , wherein the sample is additionally contacted with an aptamer, wherein the aptamer is able to specifically bind the analyte, and wherein the first and second monomers polymerize when the aptamer is bound by the analyte.