Sieving RNA 3D Structures with SHAPE and Evaluating Mechanisms Driving Sequence-Dependent Reactivity Bias

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing provides local RNA flexibility information at single-nucleotide resolution. In general, SHAPE is thought of as a secondary structure (2D) technology, but we find evidence that robust tertiary structure (3D) information is contained in SHAPE data. Here, we report a new model that achieves a higher correlation between SHAPE data and native RNA 3D structures than the previous 3D structure-SHAPE relationship model. Furthermore, we demonstrate that the new model improves our ability to discern between SHAPE-compatible and -incompatible structures on model decoys. After identifying sequence-dependent bias in SHAPE experiments, we propose a mechanism driving sequence-dependent bias in SHAPE experiments, using replica-exchange umbrella sampling simulations to confirm that the SHAPE sequence bias is largely explained by the stability of the unreacted SHAPE reagent in the binding pocket. Taken together, this work represents multiple practical advances in our mechanistic and predictive understanding of SHAPE technology.