Functional nucleic acid ligands to fluorescent proteins
The present invention relates to a nucleic acid aptamer having a first domain that binds to a fluorescent protein. The nucleic acid aptamer forms a molecular complex whereby the aptamer binds a fluorescent protein at the first domain. A constructed DNA molecule, expression systems, and host cells containing the molecular complex are also disclosed. The invention also relates to a system containing a first DNA molecule encoding the nucleic acid aptamer of the present invention and a second DNA molecule encoding a fluorescent protein capable of being bound by the first domain. Methods of detecting a molecular target and determining location of a molecular target using the nucleic acid aptamer of the invention are also disclosed.
1. A nucleic acid aptamer comprising:
a core region comprising the sequence of SEQ ID NO:19;
5′ primer region and a 3′ primer region; and
one or both of a 5′ random nucleotide sequence region and a 3′ random nucleotide sequence region, wherein the nucleic acid aptamer has a secondary structure comprising three stems and a central loop and binds to a fluorescent protein selected from GFP, eGFP, eCFP, and eYFP.
2. The nucleic acid aptamer according to claim 1 , wherein the nucleic acid aptamer reversibly binds to the fluorescent protein.
3. A molecular complex comprising the nucleic acid aptamer according to claim 1 and a fluorescent protein selected from GFP, eGFP, eCFP, and eYFP bound to the nucleic acid aptamer.
4. The molecular complex according to claim 3 , wherein the nucleic acid aptamer is reversibly bound to the fluorescent protein.
5. The molecular complex according to claim 3 , wherein the binding of the nucleic acid aptamer alters fluorescence of the fluorescent protein.
6. A host cell containing the molecular complex according to claim 3 .
7. A method of detecting a molecular target comprising:
providing the molecular complex according to claim 3 and
contacting the molecular target with the molecular complex under conditions effective to cause a change in fluorescence of the fluorescent protein, whereby the change in fluorescence detects presence of the molecular target.
8. The method according to claim 7 , wherein said contacting causes release of the fluorescent protein from the nucleic acid aptamer, an increase in fluorescence, or a decrease in fluorescence, or said contacting inhibits fluorescence.
9. The method according to claim 7 , wherein said contacting is carried out in a cell.
10. The method according to claim 9 , wherein said providing is carried out by recombinantly expressing the molecular complex in the cell.
11. A method of determining location of a molecular target comprising:
providing the molecular complex according to claim 3 ;
contacting the molecular target with the molecular complex; and
detecting the fluorescence of the fluorescent protein, whereby location of the molecular target is determined.
12. The method according to claim 11 , wherein said contacting is carried out in a cell.
13. The nucleic acid aptamer according to claim 1 comprising both the 5′ random nucleotide sequence region and the 3′ random nucleotide sequence region.
14. The nucleic acid aptamer according to claim 13 , wherein the 5′ random nucleotide sequence region, the core region, and the 3′ random nucleotide sequence region have nucleotide sequence of SEQ ID NO:22.
15. The nucleic acid aptamer according to claim 13 , wherein the 5′ random nucleotide sequence region, the core region, and the 3′ random nucleotide sequence region have the nucleotide sequence of SEQ ID NO:23.
16. The nucleic acid aptamer according to claim 13 , wherein the 5′ random nucleotide sequence region, the core region, and the 3′ random nucleotide sequence region have the nucleotide sequence of SEQ ID NO:24.
17. The nucleic acid aptamer according to claim 13 , wherein the 5′ random nucleotide sequence region, the core region, and the 3′ random nucleotide sequence region have the nucleotide sequence of SEQ ID NO:25.
18. The nucleic acid aptamer according to claim 13 , wherein the 5′ random nucleotide sequence region, the core region, and the 3′ random nucleotide sequence region have the nucleotide sequence of SEQ ID NO:26.
19. The nucleic acid aptamer according to claim 13 , wherein the 5′ random nucleotide sequence region, the core region, and the 3′ random nucleotide sequence region have the nucleotide sequence of SEQ ID NO:27.
20. The nucleic acid aptamer according to claim 13 , wherein the 5′ random nucleotide sequence region, the core region, and the 3′ random nucleotide sequence region have the nucleotide sequence of SEQ ID NO:28.
21. The nucleic acid aptamer according to claim 1 , wherein the aptamer has the nucleotide sequence of SEQ ID NO:29.