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In Vitro Directed Evolution of Fluorescence-Enhancing and Structure-Switching RNA Aptamers


Genetically-encoded fluorescent proteins have become an essential tool, and researchers today have access to a diverse array of protein-labeling strategies for studying fundamental \textit{in vivo} processes. However, there exist relatively few methods for \textit{in vivo} detection of an equally important biomolecule – RNA. One useful method to track RNA entails fusing the cellular RNA of interest to an RNA aptamer that can bind to and “turn on” the fluorescence of a small-molecule dye. Unfortunately, it is difficult to discover such fluorescence-enhancing RNA aptamers. This is because the conventional method of aptamer discovery (SELEX) can enrich for RNA that binds to a dye, but it does not preferentially identify sequences that generate a fluorescent signal upon binding. Thus, there is a critical need for a method capable of directly screening RNA aptamers for fluorescence enhancement.

To address this need, we have developed a strategy for rapidly and intently screening large numbers of RNA aptamers based on their capacity to generate a fluorescent signal upon binding a small-molecule dye. Our approach generates libraries of Gene-linked RNA Aptamer Particles (GRAPs) that display functional RNA aptamers and are isolatable using fluorescence-activated cell sorting (FACS). As proof of concept, we performed selections isolating fluorescence-enhancing aptamers against malachite green (MG). We show that by directly selecting for function rather than binding, we can isolate RNA aptamers that are brighter and higher affinity than the best known MG aptamer. GRAP display also enables us to measure the fluorescence signal of every aptamer across multiple emission windows, permitting us to reconstruct the emission profile of every displayed aptamer. This in turn, allows us to intentionally isolate aptamers that fluoresce at a variety of wavelengths upon binding their target dye. Lastly, this technique can be used to discover functional RNA that undergoes a structural re-organization upon ligand binding. This has promising implications in the fields of biosensing, gene therapy, and cellular computing. This flexibility should greatly expand the toolbox of reagents available for studying RNA in vivo.

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