In Vitro Analysis of Riboswitch-Spinach Aptamer Fusions as Metabolite-Sensing Fluorescent Biosensors

The development of fluorescent biosensors has been motivated by the interest to monitor and measure the levels of specific metabolites in live cells in real time. Common approaches include fusing a protein-based receptor to fluorescent proteins or synthesizing a small molecule reactive probe. Natural metabolite-sensing riboswitches also have been used in reporter-based systems that take advantage of ligand-dependent regulation of downstream gene expression. More recently, it has been shown that RNA-based fluorescent biosensors can be generated by fusing a riboswitch aptamer to the in vitro selected Spinach aptamer, which binds a cell-permeable and conditionally fluorescent molecule. Here, we describe methods to design, prepare, and analyze riboswitch-Spinach aptamer fusion RNAs for ligand-dependent activation of fluorescence in vitro. Examples of procedures to measure fluorescence activation, ligand binding selectivity and affinity, and binding kinetics are given for a cyclic di-GMP-responsive biosensor. The relative ease of in vitro RNA synthesis and purification should make this method accessible to other researchers interested in developing riboswitch-based fluorescent biosensors.