Visualization and Quantification of Intermolecular RNA Base Pairing in in vitro RNA Clusters Using Split Broccoli RNA...

RNA clustering, driven by multivalent intermolecular RNA-RNA interactions, can occur in vitro in the absence of proteins. While chemical probing and computational simulations have been used to predict these interactions, methods for directly assessing intermolecular base pairing in RNA clusters remain limited. This study presents a visual assay based on split Broccoli RNA reporters conjugated to fluorescently labeled RNAs of interest. The assay enables the detection and quantification of intermolecular base pairing in RNA clusters. The split Broccoli system contains complementary reporter sequences that dimerize to form the Broccoli RNA aptamer. The resulting dimerized RNA structure binds DFHBI-1T, a fluorophore that emits green fluorescence upon binding. Upon RNA clustering, the appearance of green fluorescence reports base pairing mediated by the complementary reporter sequences among the RNAs of interest. Measurement of DFHBI-1T fluorescence enables quantitative assessment of how in vitro RNA clustering conditions influence intermolecular base pairing between these reporter sequences.