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RNA imaging paves the way to investigate RNA function and regulation from the aspect of RNA subcellular localization and expression level. In the past decades, various highly precise and genetic coding fluorescent RNA, which can bind and light up the fluorophore by their specific sequence/structure, has been developed for RNA imaging, including Broccoli, Mango, SRB‐2, RhoBAST, and Pepper et al. In this Review, we summarize the RNA imaging approaches and the progress made in the development of fluorescent RNA. Next, we emphasize the application of fluorescent RNA for in situ RNA imaging in living cells. Of particular note, besides the artificially selected fluorescent RNAs, a new kind of fluorophore that can bind and light up intrinsic RNA structure is described together for the first time. Finally, a summary of current strategies for designing fluorescent RNA and future perspectives are also presented. We hope this expanding topic will inspire the study of the architecture and functions of these fascinating molecular machinery in biological systems. [ABSTRACT FROM AUTHOR]

Fluorescent light-up aptamers (FLAPs) are well-performed biosensors for cellular imaging and the detection of different targets of interest, including RNA, non-nucleic acid molecules, metal ions, and so on. They could be easily designed and emit a strong fluorescence signal once bound to specified fluorogens. Recently, one unique aptamer called Mango-II has been discovered to possess a strong affinity and excellent fluorescent properties with fluorogens TO1-Biotin and TO3-Biotin. To explore the binding mechanisms, computational simulations have been performed to obtain structural and thermodynamic information about FLAPs at atomic resolution. AMOEBA polarizable force field, with the capability of handling the highly charged and flexible RNA system, was utilized for the simulation of Mango-II with TO1-Biotin and TO3-Biotin in this work. The calculated binding free energy using published crystal structures is in excellent agreement with the experimental values. Given the challenges in modeling complex RNA dynamics, our work demonstrates that MD simulation with a polarizable force field is valuable for understanding aptamer-fluorogen binding and potentially designing new aptamers or fluorogens with better performance.

The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light‐up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell‐permeable small‐molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP‐tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA–RNA and RNA–protein interactions using split‐FLAP and Förster resonance energy transfer (FRET) systems is given. Bright future for FLAPs: Fluorescent light‐up aptamers (FLAPs) are genetically encodable RNA sequences that fluoresce upon binding of small‐molecule fluorogens. These are increasingly being developed into systems for the investigation of cellular RNAs. In this Minireview, the potential of FLAPs in the study of bimolecular RNA–RNA or RNA–protein interactions is explored. [ABSTRACT FROM AUTHOR]