Samie Jaffrey

Imaging RNA and RNA biology using RNA mimics of green fluorescent protein

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 until they are bound by these RNAs.  These fluorescent RNA-fluorophore complexes 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 can be fused to RNAs and expressed in cells in order to image RNA localization and other RNA regulatory processes in cells. Spinach can be used to create sensors composed of RNA that enable the imaging of other biological molecules. We will discuss novel structure-guided and directed evolution approaches for generating novel optimized Spinach variants with improved photophysical properties.  We have used these approaches, as well as new fluorophore chemistries to create new RNA-fluorophore complexes suitable for imaging in yellow and red fluorescence channels.  These RNA mimics of GFP provide novel approaches to image RNA biology and other processes in cells.