Combining Biophysical Methods for Structure-Function Analyses of RNA in Solution.

Abstract

RNA-level regulation by riboswitches relies on the specific binding of small metabolites to the aptamer domain to trigger substantial conformational changes that affect transcription or translation. Although several biophysical methods have been employed to study such RNAs, the utility of any one single method is limited. Hybrid approaches, therefore, are essential to better characterize these intrinsically dynamic molecules and elucidate their regulatory mechanisms driven by ligand-induced conformational changes. This chapter outlines procedures for biochemical and biophysical characterization of RNA that employs a combination of solution-based methods: isothermal titration calorimetry (ITC), small-angle X-ray scattering (SAXS), and atomic force microscopy (AFM). Collectively, these tools provide a semi-quantitative assessment of the thermodynamics associated with ligand binding and subsequent conformational changes.