S134: Imaging intracellular metabolites with RNA mimics of green fluorescent protein

The major tools for imaging small molecules in real time are genetically encoded reporters, which are typically composed of two fluorescent proteins that undergo conformational changes upon ligand binding, which is detected by changes in FRET.  However, sensors for most small molecules are difficult to generate since the appropriate protein which binds and undergoes a conformational change is not available.  We have developed a novel class of fluorescent indicators of small molecules that are composed of RNA.  This approach takes advantage of the ease with which RNA aptamers can be generated which can bind target molecules with high specificity and affinity.  These RNA sensors comprise a target-binding RNA sequence and the Spinach sequence, which binds and switches on the fluorescence of a small molecule fluorophore.  We have shown these sensors can be used to detect a variety of different small molecules with high selectivity and affinities which correlates with physiological levels of these metabolites.  Furthermore, we show that these sensors can be used to monitor dynamic changes and cell-to-cell variation in the intracellular levels of adenosine 5’-diphosphate (ADP) and S-adenosylmethionine (SAM), two important signaling molecules in E. coli.  This novel and versatile approach for generating sensors has the potential to generate sensors for essentially any intracellular signaling molecule and can be used to monitor or optimize microbial production of biomolecules in bacterial cells.