Mapping RNA Structure In Vitro Using Nucleobase-Specific Probes

RNAs have to adopt specific three-dimensional structures to fulfill their biological functions. Therefore exploring RNA structure is of interest to understand RNA-dependent processes. Chemical probing in vitro is a very powerful tool to investigate RNA molecules under a variety of conditions. Among the most frequently used chemical reagents are the nucleobase-specific probes dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT) and β-ethoxy-α-ketobutyraldehyde (kethoxal). These chemical reagents modify nucleotides which are not involved in hydrogen bonding or protected by a ligand, such as proteins or metabolites. Upon performing modification reactions with all three chemicals the accessibility of all four nucleobases can be determined. With this fast and inexpensive method local changes in RNA secondary and tertiary structure, as well as the formation of contacts between RNA and its ligands can be detected independent of the RNA’s length.