Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

RNA function is determined by its structural organization. The RNA structure consists of the combination of distinct secondary structure motifs connected by junctions that play an essential role in RNA folding. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probing is an established methodology to analyze the secondary structure of long RNA molecules in solution, which provides accurate data about unpaired nucleotides. However, the residues located at the junctions of RNA structures usually remain undetected. Here we report an RNA probing method based on the use of a novel open-paddlewheel diruthenium (OPW-Ru) compound Ru₂Cl₂(µ-DPhF)₃(DMSO)] (DPhF = N,N′-diphenylformamidinate). This compound has four potential coordination sites in a singular disposition to establish covalent bonds with substrates. As a proof of concept, we have analyzed the reactivity of OPW-Ru toward RNA using two viral internal ribosome entry site (IRES) elements whose function depends on the structural organization of the molecule. Our study suggests that the compound OPW-Ru preferentially attacks at positions located one or two nucleotides away from junctions or bulges of the RNA structure. The OPW-Ru fingerprinting data differ from that obtained by other chemical reagents and provides new information about RNA structure features.