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 of forming a structure comprising hybridized nucleic acid monomers, the method comprising:
a) providing an initiator comprising a nucleic acid initiation region and first complementary region;
b) providing 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 the first complementary region of the initiator,
ii) a first hairpin loop region at an end of the first duplex region, wherein said first hairpin loop region is closed by a single base pair, and
iii) an initiator complement region that comprises a sticky end and that is substantially complementary to the initiation region of the initiator;
(c) providing 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;
wherein the first and second monomers hybridize in a chain reaction upon binding of the initiator to the first monomer.
2. The method of claim 1 , wherein the first loop region comprises from about 3 to about 30 nucleotides.
3. The method of claim 1 , wherein the initiator is an analyte to be detected in a sample.
4. The method of claim 1 , wherein the initiator is a single stranded nucleic acid.
5. The method of claim 1 , wherein the initiator additionally comprises a recognition molecule.
6. The method of claim 5 , wherein the recognition molecule is an aptamer.
7. The method of claim 1 , wherein the initiator undergoes a conformational change in the presence of an analyte.
8. The method of claim 1 , wherein the structure comprising hybridized monomers is detected by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, mass spectrometry, light scattering spectroscopy, colorimetry and fluorescent spectroscopy.
9. The method of claim 1 , further comprising:
providing a third nucleic acid monomer comprising a sixth complementary region that is complementary to a portion of the first and/or second monomer; and
providing a fourth nucleic acid monomer comprising a seventh complementary region that is complementary to a portion of the third monomer.
10. The method of claim 9 , wherein the first monomer comprises a bulge region that is complementary to a portion of the third monomer.
11. 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.
12. A method of forming a structure comprising hybridized nucleic acid monomers, the method comprising:
a) providing an initiator comprising a nucleic acid initiation region and first complementary region;
b) providing 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 the first complementary region of the 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 the initiation region of the initiator;
(c) providing 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;
wherein the first and second monomers hybridize in a chain reaction upon binding of the initiator to the first monomer.
13. The method of claim 12 , wherein the first loop region comprises from about 3 to about 30 nucleotides.
14. The method of claim 12 , wherein the initiator is an analyte to be detected in a sample.
15. The method of claim 12 , wherein the initiator is a single stranded nucleic acid.
16. The method of claim 12 , wherein the initiator additionally comprises a recognition molecule.
17. The method of claim 16 , wherein the recognition molecule is an aptamer.
18. The method of claim 12 , wherein the initiator undergoes a conformational change in the presence of an analyte.
19. The method of claim 12 , wherein the structure comprising hybridized monomers is detected by a method selected from the group consisting of gel electrophoresis, capillary electrophoresis, mass spectrometry, light scattering spectroscopy, colorimetry and fluorescent spectroscopy.
20. The method of claim 12 , further comprising:
providing a third nucleic acid monomer comprising a sixth complementary region that is complementary to a portion of the first and/or second monomer; and
providing a fourth nucleic acid monomer comprising a seventh complementary region that is complementary to a portion of the third monomer.
21. The method of claim 20 , wherein the first monomer comprises a bulge region that is complementary to a portion of the third monomer.
22. The method of claim 12 , 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.