Fingerprinting Tertiary Structure in Complex RNAs Using Single-Molecule Correlated Chemical Probing

Single-molecule correlated chemical probing (smCCP) is an experimentally concise strategy for characterizing higher-order structural interactions in RNA. smCCP data yield rich, but complex, information about base pairing, conformational ensembles, and tertiary interactions. To date, through-space communication specifically measuring RNA tertiary structure has been difficult to isolate from structural communication reflective of other interactions. Here, we introduce mutual information as a filtering metric to isolate tertiary structure communication contained within smCCP data and use this strategy to characterize the structural ensemble of the SAM-III riboswitch. We identified an smCCP fingerprint that is selective for states containing a tertiary structure that forms concurrently with cognate ligand binding. We then successfully applied mutual information filters to independent RNAs and isolated through-space tertiary interactions in riboswitches and large RNAs with complex structures. smCCP, coupled with mutual information criteria, can now be used as a tertiary structure discovery tool, including to identify specific states in an ensemble that have a higher-order structure. These studies pave the way for the use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs.