Methods for identifying aptamer regulators
Methods are provided for identifying aptamer regulators. Aptamer regulators are aptamers that bind to a target wherein binding of the aptamer regulator to the target increases the binding affinity of the target for a target partner relative to the affinity of the target for the target partner when the target is not bound by the aptamer regulator such that binding of the aptamer regulator to the target is a prerequisite for target-target partner complex formation.
1. A method for identifying an aptamer regulator comprising the steps:
a) providing a target and a target partner that do not bind to each other in the absence of an aptamer regulator;
b) contacting a mixture of nucleic acids with the target partner under conditions that favor specific binding between the nucleic acids and the target partner;
c) partitioning the bound nucleic acids from the unbound nucleic acids, and retaining the unbound nucleic acids;
d) contacting the unbound nucleic acids with the target and the target partner;
e) partitioning nucleic acids bound to a target-target partner complex from unbound nucleic acids, wherein binding of a nucleic acid to the target induces a conformational change in the target that increases the binding affinity of the target for the target partner relative to when the target is not bound by the nucleic acid;
f) retaining the nucleic acids bound to the target-target partner complex; and
g) removing the retained nucleic acids that are bound to the target in the target-target partner complex,
thereby identifying an aptamer regulator that binds to the target and induces a conformational change in the target that increases the binding affinity of the target for the target partner relative to the affinity of the target for the target partner without the aptamer regulator, wherein the binding of the aptamer regulator to the target is a prerequisite for target-target partner complex formation.
2. The method of claim 1 , wherein the mixture of nucleic acids is a target-specific pool of nucleic acids having high affinity and specificity for the target.
3. The method of claim 2 , wherein the target-specific pool of nucleic acids is diversified.
4. The method of claim 1 , wherein the target partner is immobilized.
5. The method of claim 1 , wherein the removing is by eluting the nucleic acids with an agonist competitor to the target.
6. The method of claim 1 , wherein the removing is by contacting the bound nucleic acids with excess free target.
7. The method of claim 1 , wherein the method further comprises the step of amplifying the retained nucleic acids and repeating steps a) to b).
8. The method of claim 1 , wherein the method further comprises the step of screening the nucleic acids retained in step d) for a desired functional activity.