Abstract
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.
Highlights
The fact that RNA folding and the RNA interactome in viral genomes are well conserved makes it tempting to propose them as potential targets for the development of new antiviral treatments [4]
This article describes in detail protocols focused on the application of high-throughput probing methods followed in our laboratory to decipher RNA-RNA interactions in viral genomes
After the purification of the different RNA conformers, nucleotide modification is detected by primer extension, as described for the Dimethyl sulfate (DMS)
Summary
The siRNA technology, which has been extensively tested against a wide variety of viruses (for a review see [5]) These studies have proved that the efficiency of different siRNAs against the same target gene can be different; confirming that the random design of siRNAs is not a valid strategy when it comes to obtaining efficient inhibitors. The biochemical and biophysical study of RNA folding, together with the use of bioinformatics strategies must be accomplished to design efficient RNA-based inhibitors This will provide an excellent starting point for the development of antiviral strategies, which is a real need for many RNA viruses that are responsible for major human diseases today. This article describes in detail protocols focused on the application of high-throughput probing methods followed in our laboratory to decipher RNA-RNA interactions in viral genomes
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