Trimodular Photoprobes – Isolation of small-molecule binding RNA by photoaffinity crosslinking

While photoaffinity crosslinking has been successfully used to probe protein-protein and protein-small molecule interactions, it is only scarcely utilized in studies involving RNA. Here we combine the use of photoaffinity crosslinking with ligation mediated amplification techniques to isolate and characterize even transiently interacting unknown RNA sequences for a given small molecule.

Our photoprobes are equipped with a photoactivatable aryl azide for crosslinking, a terminal alkyne to attach it to a reporter tag or an affinity probe by copper-catalyzed click chemistry and a small molecule whose target needs to be found. After being bound to its target RNA, the photoprobe is covalently crosslinked to its target by UV irradiation. The resulting covalently linked species are purified by affinity chromatography. The following adapter ligation introduces constant primer binding sites, therefore allowing us to characterize the unknown RNAs. We have developed a universal ligation procedure to achieve quantitative ligation of adapters to any unknown, chemically lesioned, immobilized RNA of choice irrespective of its length and structural complexity.

The photoaffinity probes used for this study are synthesized via a convergent synthetic route leading to a small library of photoprobes for a broad spectrum of small molecules ranging from amino acids like lysine or glycine, Nature’s methylating agent SAM, energy carriers like ATP to antibiotics like Trimethoprim.