title: Developing Fluorogenic Riboswitches for Imaging Metabolite Concentration Dynamics in Bacterial Cells.
abstract: Genetically encoded small-molecule sensors are important tools for revealing the dynamics of metabolites and other small molecules in live cells over time. We recently developed RNA-based sensors that exhibit fluorescence in proportion to a small-molecule ligand. One class of these RNA-based sensors are termed Spinach riboswitches. These are RNAs that are based on naturally occurring riboswitches, but have been fused to the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, producing fluorescence upon binding the cognate small-molecule analyte. Here, we describe how to design and optimize these sensors by adjusting critical sequence elements, guided by structural insights from the Spinach aptamer. We provide a stepwise procedure to characterize sensors in vitro and to express sensors in bacteria for live-cell imaging of metabolites. Spinach riboswitch sensors offer a simple method for fluorescence measurement of a wide range of metabolites for which riboswitches exist, including nucleotides and their derivatives, amino acids, cofactors, cations, and anions.
authors.affiliation: Tri-Institutional Chemical Biology Program at Weill-Cornell Medical College, Rockefeller University, Memorial Sloan-Kettering Cancer Center, New York, NY, United States; Weill Medical College, Cornell University, New York, NY, United States.
authors.forename: J L
authors.initials: JL
authors.lastname: Litke
authors.affiliation: Weill Medical College, Cornell University, New York, NY, United States.
authors.forename: M
authors.initials: M
authors.lastname: You
authors.affiliation: Tri-Institutional Chemical Biology Program at Weill-Cornell Medical College, Rockefeller University, Memorial Sloan-Kettering Cancer Center, New York, NY, United States; Weill Medical College, Cornell University, New York, NY, United States. Electronic address: srj2003@med.cornell.edu.
authors.forename: S R
authors.initials: SR
authors.lastname: Jaffrey
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001419:Bacteria / Q000166:cytology / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D005470:Fluorometry / Q000379:methods; D008958:Models, Molecular; D058928:Riboswitch; D018724:Spinacia oleracea / Q000235:genetics*
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D058928:Riboswitch
keywords: Cellular imaging; Dynamics; Fluorescence; Metabolite; RNA probes; Riboswitch; Sensor