Protocol for detecting in vitro riboswitch conformational switching using a fluorescence anisotropy single-stranded RNA-targeting approach

We present a high-sensitivity fluorescence anisotropy-based protocol (fluorescence anisotropy single-stranded targeting [FASST]) to monitor in vitro ligand-induced conformational switching of riboswitches. We first describe the sequence design of single-stranded fluorescent DNA probes that selectively bind to specific riboswitch conformational states. Next, we describe DNA probe binding affinity and conformational switching measurements using a 96-well plate reader. We then detail the procedures for data analysis. This protocol has the potential to screen novel riboswitch ligands based on their capacity to target functional conformational changes. For complete details on the use and execution of this protocol, please refer to Rivera et al.(1).