<i>Ab initio</i> prediction of RNA structure ensembles with RNAnneal

RNA utilizes three-dimensional structure in addition to sequence to carry out diverse functions in gene expression and disease. Much like well-folded proteins, RNAs adopt specific three-dimensional structures to carry out their function. Yet comparatively few RNA structures have been solved by atomic resolution structural techniques, in part because unlike structured proteins, RNAs fold into heterogeneous ensembles of interconverting structures that pose a challenge for high-resolution structure probing methods. In this work, we introduce RNAnneal as a method for RNA structural ensemble prediction that seamlessly integrates generative deep learning with statistical physics and molecular dynamics modeling. Given the primary sequence, RNAnneal uses <i>ab inito</i> (i.e., first principles) modeling to sample an ensemble of 3D structures, which, in turn, are used to train an ensemble of unsupervised deep learning models. The RNAnneal score, representing the consensus of the deep learning models, is then used to evaluate the 3D structures. We evaluated RNAnneal structures against 16 experimentally-resolved conformations (ERCs) of riboswitch RNAs and found that pseudoknot-free (PK-free) ERCs were well-reproduced by RNAnneal, with clear avenues for improving performance even on PK-comprising structures. Furthermore, we found that the RNAnneal score outperforms the Rosetta score and a state-of-the-art RNA forcefield on the task of classifying ERCs from decoys. We then introduce the interaction entropy as a measure of conformational heterogeneity within an ensemble and use it to assess our predictions. RNAnneal thus provides a generalizable framework for predicting RNA structural ensembles that will accelerate RNA-targeted drug discovery and the design of functional RNA molecules.