Comparison of Three Computational Tools for the Prediction of RNA Tertiary Structures

Understanding the structures of noncoding RNAs (ncRNAs) is important for the development of RNA-based therapeutics. There are inherent challenges in employing current experimental techniques to determine the tertiary (3D) structures of RNAs with high complexity and flexibility in folding, which makes computational methods indispensable. In this study, we compared the utilities of three advanced computational tools, namely RNAComposer, Rosetta FARFAR2, and the latest AlphaFold 3, to predict the 3D structures of various forms of RNAs, including the small interfering RNA drug, nedosiran, and the novel bioengineered RNA (BioRNA) molecule showing therapeutic potential. Our results showed that, while RNAComposer offered a malachite green aptamer 3D structure closer to its crystal structure, the performances of RNAComposer and Rosetta FARFAR2 largely depend upon the secondary structures inputted, and Rosetta FARFAR2 predictions might not even recapitulate the typical, inverted "L" shape tRNA 3D structure. Overall, AlphaFold 3, integrating molecular dynamics principles into its deep learning framework, directly predicted RNA 3D structures from RNA primary sequence inputs, even accepting several common post-transcriptional modifications, which closely aligned with the experimentally determined structures. However, there were significant discrepancies among three computational tools in predicting the distal loop of human pre-microRNA and larger BioRNA (tRNA fused pre-miRNA) molecules whose 3D structures have not been characterized experimentally. While computational predictions show considerable promise, their notable strengths and limitations emphasize the needs for experimental validation of predictions besides characterization of more RNA 3D structures.