In Vitro Methods to Decipher the Structure of Viral RNA Genomes

RNA viruses encode essential information in their genomes as conserved structural elements that are involved in efficient viral protein synthesis, replication, and encapsidation. These elements can also establish complex networks of RNA-RNA interactions, the so-called RNA interactome, to shape the viral genome and control different events during intracellular infection. In recent years, targeting these conserved structural elements has become a promising strategy for the development of new antiviral tools due to their sequence and structural conservation. In this context, RNA-based specific therapeutic strategies, such as the use of siRNAs have been extensively pursued to target the genome of different viruses. Importantly, siRNA-mediated targeting is not a straightforward approach and its efficiency is highly dependent on the structure of the target region. Therefore, the knowledge of the viral structure is critical for the identification of potentially good target sites. Here, we describe detailed protocols used in our laboratory for the in vitro study of the structure of viral RNA genomes. These protocols include DMS (dimethylsulfate) probing, SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) analysis, and HMX (2'-hydroxyl molecular interference). These methodologies involve the use of high-throughput analysis techniques that provide extensive information about the 3D folding of the RNA under study and the structural tuning derived from the interactome activity. They are therefore a good tool for the development of new RNA-based antiviral compounds.