RNAs have highly complex and dynamic cellular localization patterns. Technologies for imaging RNA in living cells are important for uncovering their function and regulatory pathways. One approach for imaging RNA involves genetically encoding fluorescent RNAs using RNA mimics of green fluorescent protein (GFP). These mimics are RNA aptamers that bind fluorophores resembling those naturally found in GFP and activate their fluorescence. These RNA–fluorophore complexes, including Spinach, Spinach2, and Broccoli, can be used to tag RNAs and to image their localization in living cells. In this article, we describe the generation and optimization of these aptamers, along with strategies for expanding the spectral properties of their associated RNA–fluorophore complexes. We also discuss the structural basis for the fluorescence and photophysical properties of Spinach, and we describe future prospects for designing enhanced RNA–fluorophore complexes with enhanced photostability and increased sensitivity.


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