Abstract
Fluorescence anisotropy provides a powerful method to quantitatively characterize kinetic and thermodynamic parameters for many RNA–ligand interactions. By manipulating assay design, it is possible to extract equilibrium binding constants down to the low or even sub-nanomolar range, as well as measure reaction on- and off-rates, changes in enthalpy and entropy, and RNA-binding site size. These tools are invaluable for comparing and contrasting potential mechanisms of RNA recognition by proteins or other binding partners. In this chapter, we review the theory behind the technique and describe some recent applications. We explain how to generate binding isotherms using fluorescence anisotropy, including discussion of the instrumentation and fluorescent substrates required. To analyze isotherms, we derive relationships between anisotropy and free protein concentration for a variety of reaction mechanisms and discuss statistical validation methods. Finally, we have included sections on the use of plate readers for measuring fluorescence anisotropy across large numbers of samples and their application to high-throughput drug screening. Fluorescence anisotropy has been used to elucidate mechanisms involved in many critical processes in RNA biology, including assembly of the translation initiation complex and recognition of mRNAs targeted for degradation.