RNA-Based Fluorescent Biosensors for Detecting Metabolites in vitro and in Living Cells.

Abstract

Genetically encoded sensors are important tools for measuring metabolites and other small molecules in vitro and in live cells. Until recently, genetically encoded sensors exclusively comprised fluorescent proteins that undergo changes in Förster resonance energy transfer upon binding a target analyte. However, recently a new class of fluorescent sensor has been developed composed of RNA. These RNA-based sensors rely on Spinach and other RNA mimics of green fluorescent protein. In each case, the RNA-based sensors contain an analyte-binding aptamer domain which transduces binding of the analyte into a conformational change in Spinach. Two types of sensors have been developed: allosteric Spinach sensors and Spinach riboswitches. Allosteric Spinach sensors exhibit metabolite-induced folding and subsequent fluorescence. Spinach riboswitches are naturally occurring riboswitches that have been modified to contain the Spinach aptamer. The resulting RNA is a fluorogenic riboswitch, and produces fluorescence upon binding its cognate analyte. We describe the development of this new technology, its uses, and future directions to facilitate the use of this assay technology in mammalian cells and in high-throughput applications.