RNA analogs of green fluorescent protein for live cell RNA imaging and development of small molecule sensors

Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various in vitro and in vivo applications, but a comparably robust and simple approach for fluorescently tagging RNA is lacking. We now describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNAs activate the fluorescence of these fluorophores, resulting in a palette of RNA-fluorophore complexes that span the visible spectrum. An RNA-fluorophore complex resembling enhanced GFP (EGFP), termed Spinach, emits a green fluorescence comparable in brightness to fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach to genetically encode fluorescent RNAs. Additionally, we have shown that these RNA aptamers can be fused to other aptamers that bind to biologically relevant metabolites to generate allosterically regulated fluorescent metabolite sensors. Development of these RNA sensors will yield the generation of genetically encodable fluorescent sensors to detect endogenous small molecules, metabolites, proteins, lipids, nucleic acids, or other potentially important biological molecules. Moreover, these sensors could be developed as a new fluorescent sensor platform for in vitro applications.