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RNA mimics of fluorescent proteins: new tools for the in vivo study of noncoding RNAs

Adrian R. Ferré-D'Amaré Laboratory of RNA Biophysics and Cellular Physiology National Heart, Lung and Blood Institute Bethesda, MD 20892-8012

Recently, RNA molecules that induce the fluorescence of small molecule ligands by more than 1000-fold and can be used as genetically encoded tags for in vivo imaging of RNAs have been described. These molecules have the potential to revolutionize the cell biology RNAs much as green fluorescent protein (GFP) and its variants have transformed studies of the proteome. We have determined crystal structures of RNAs that fluoresce green, yellow and orange (named Spinach, Corn and Mango, respectively) and discovered that they have unrelated overall folds, but are similar in employing a G-quadruplex as a platform to bind their respective fluorophores. The dissimilar folds of these fluorogenic RNAs contrast with the structures of fluorescent proteins, which are largely variations of one fold (the celebrated 11-stranded β-barrel). Importantly, the divergent structures of in vitro selected fluorogenic RNAs translate to diverse biochemical and biophysical properties. For instance, Spinach loses fluorescence rapidly upon illumination, but can rebind to fresh fluorophore, and therefore can be fluorescent indefinitely. Corn and Mango are more photostable, but cannot bind to fresh fluorophore once they bleach. Mango has very high affinity (Kd ~ 3 nM) for its fluorophore, and with a suitable fluorophore is more red-shifted than the protein mPlum. Corn is an obligate asymmetric dimer that binds to one fluorophore molecule, and could thus serve as the basis for development of RNA analogs of split GFP. The structural diversity of fluorogenic RNAs, coupled with the chemical diversity of small molecule fluorophores suitable for binding to RNA has the potential to yield a large toolkit for RNA cell biology wherein specific RNA/fluorophore pairs will be optimized for different experimental needs. 1. Trachman, R.J. III, Truong, L., & Ferré-D'Amaré, A.R. Structural principles of fluorescent RNA aptamers. Trends Pharmacol. Sci. 38, 928-939 (2017). 2. Trachman, R.J. III, Demeshkina, N.A., Lau, M.W.L., Panchapakesan, S.S.S., Jeng, S.C.Y., Unrau, P.J., & Ferré-D'Amaré, A.R. Structural basis for high-affinity fluorophore binding and activation by RNA Mango. Nature Chem. Biol. 13, 807-813 (2017). 3. Warner, K.D., Sjekloca, L., Song, W., Filonov, G.S., Jaffrey, S.R. & Ferré-D'Amaré, A.R. A homodimer interface without base pairs in an RNA mimic of red fluorescent protein. Nature Chem. Biol. 13, 1195-1201 (2017). 4. Warner, K.D., Chen, M.C., Song, W., Strack, R.L., Thorn, A., Jaffrey, S.R., & Ferré-D'Amaré, A.R. Structural basis for activity of highly efficient RNA mimics of green fluorescent protein. Nature Struct. Mol. Biol. 21, 658-663 (2014).