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, structural information on 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 a smCCP fingerprint that is selective for states containing 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 higher-order structure. These studies pave the way for use of the straightforward smCCP experiment for discovery and characterization of tertiary structure motifs in complex RNAs. In-vitro transcribed SAM-III riboswitch constructs with structure cassettes, either native sequence or 1 of 4 structure-perturbing mutants, were refolded and structure was probed with DMS (or non-reactive control). Modified RNA was reverse transcribed under MaP conditions using SuperScript II. cDNA sequencing libraries were prepared and sequenced on an Illumina MiSeq instrument.