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 • Instructions for design of conformationally selective fluorescent DNA probes • Steps for probe affinity evaluation • Procedures for switching constant determination Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.
Clairoux et al. (Thu,) studied this question.