Visualizing RNA conformational and architectural heterogeneity in solution

Abstract RNA flexibility is reflected in its heterogeneous conformation. Existing techniques, such as NMR, crystallography, or cryo-EM, are not suited for studying highly heterogeneous conformation. Through direct visualization using atomic force microscopy (AFM) and the adenosylcobalamin riboswitch aptamer domain as an example, we show that a single RNA sequence folds into conformationally and architecturally heterogeneous structures under near-physiological solution conditions. Recapitulated 3D topological structures from AFM molecular surfaces reveal that all conformers share the same secondary structural elements. Only a population-weighted cohort, not any single conformer, including the crystal structure, can account for the ensemble behaviors. All conformers, except for one, are functionally active in terms of ligand binding. Our findings provide direct visual evidence that the sequence-structure relationship of RNA under physiologically relevant solution conditions is more complex than the one-to-one relationship for well-structured proteins. The direct visualization of conformational and architectural ensembles in solution may prompt a revision to our current views and approaches to RNA structural analyses.