Fluorescence Anisotropy Applied to Measure Aptamer-Protein Interactions

This chapter describes a fluorescence anisotropy-based method for quantifying the binding affinity between a nucleic acid aptamer and its protein target, using the thrombin-binding aptamer (TBA)-thrombin interaction as an example. Fluorescence anisotropy is a rapid and sensitive technique that monitors changes in a molecule's rotational speed upon binding. By fluorescently labeling an aptamer and titrating it with increasing concentrations of its target protein, users can generate a binding isotherm and determine the dissociation constant (K_D) with accuracy. The protocol includes detailed instructions for reagent preparation, plate setup, data acquisition, and analysis, with emphasis on minimizing experimental variability. Although demonstrated for the thrombin aptamer system, the approach is broadly applicable to other protein-nucleic acid interactions.