Probing of Fluorogenic RNA Aptamers via Supramolecular Förster Resonance Energy Transfer with a Universal Fluorescent Nucleobase Analog

Abstract

Fluorogenic RNA aptamers are synthetic RNAs that have been evolved by in vitro selection methods to bind and light up conditionally fluorescent organic ligands. Compared with other probes for RNA detection, they are less invasive than hybridization-based methods (FISH, molecular beacons) and are considerably smaller than fluorescent protein-recruiting systems (MS2, Pumilio variants). Fluorogenic aptamers have therefore found widespread use as genetically encodable tags for RNA detection in live cells and have also been used in combination with riboswitches to construct versatile metabolite sensors for in vitro use. Their success builds on a fundamental understanding of their three-dimensional structure to explain the mechanisms of ligand interaction and to rationally design functional aptamer devices. In this protocol, we describe a supramolecular FRET-based structure probing method for fluorogenic aptamers that exploits distance- and orientation-dependent energy transfer efficiencies between site-specifically incorporated fluorescent nucleoside analogs and non-covalently bound ligands, exemplified by 4-cyanoindol riboside (4CI) and the DMHBI⁺-binding RNA aptamer Chili. This method yields structural restraints that bridge the gap between traditional low-resolution secondary structure probing methods and more elaborate high-resolution methods such as X-ray crystallography and NMR spectroscopy.