Genetically Encoded RNA Devices for Imaging RNA Biology and Cellular Signaling

Abstract: 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 will describe the creation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These fluorophores are nonfluorescent in vitro and in cells, but become fluorescent upon binding specific aptamers. A variety of fluorophores have been designed that mimic fluorophores naturally found in GFP and red fluorescent protein (RFP), and the corresponding RNA-fluorophore complexes, termed Spinach, Corn, and Red Broccoli, exhibit green, yellow and red fluorescence. These aptamers function as genetically encodable imaging tags, but also enable visualization of other cellular processes such as real-time imaging of transcription in single cells. Another use of these aptamers is for the creation of genetically encoded sensors composed of RNA that report on the endogenous metabolite and protein levels. We will discuss the various strategies for the design of these sensors as well as novel structure-guided, directed evolution, and massively parallel biochemical approaches for optimizing the use of these RNA-based sensors.