title: In vitro analysis of riboswitch-Spinach aptamer fusions as metabolite-sensing fluorescent biosensors.
abstract: 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California, USA.
authors.forename: Colleen A
authors.initials: CA
authors.lastname: Kellenberger
authors.affiliation: Department of Chemistry, University of California, Berkeley, California, USA; Department of Molecular & Cell Biology, University of California, Berkeley, California, USA. Electronic address: mingch@berkeley.edu.
authors.forename: Ming C
authors.initials: MC
authors.lastname: Hammond
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D008856:Microscopy, Fluorescence; D058928:Riboswitch / Q000235:genetics*
chemical_list: D058928:Riboswitch
keywords: Cyclic di-GMP; Cyclic dinucleotide; Fluorescence imaging; Fluorescence microscopy; RNA biosensor; Spinach aptamer; Vc2 riboswitch