Kuehrova, P. | Search bioRxiv

bioRxiv2026

Toward Accurate RNA Folding Thermodynamics: Evaluation of Enhanced Sampling Methods for Force Field Benchmarking

Biologically functional RNAs operate near marginal stability, and their rugged free-energy landscapes and profound structural dynamics - typically not captured by structural biology experiments - play decisive roles. Atomistic molecular dynamics (MD) simulations provide a unique means to characterize these features. However, the applicability of atomistic MD is currently limited by accessible simulation timescales and, most importantly, by force-field (FF) accuracy. Folding free energies ({Delta}G{degrees}fold) of small RNA motifs represent well-defined targets for quantitative benchmarking of RNA FFs. In practice, however, obtaining thermodynamic estimates that are sufficiently robust for direct comparison with experimental data remains highly challenging, even for small RNA systems, and many published studies rely on sampling that is not fully converged. Here, we systematically assess the performance of widely used advanced enhanced-sampling techniques using the 8-mer r(gcGAGAgc) tetraloop as a representative benchmark system. We test temperature replica exchange (T-REMD), two solute-tempering variants of replica exchange (REST2 and REHT), as well as well-tempered metadynamics and on-the-fly probability enhanced sampling combined with solute tempering (ST-MetaD and ST-OPES). Among the tested approaches, T-REMD proves to be the most robust, yielding reproducible folding equilibria and consistent estimates of {Delta}G{degrees}fold after approximately 20 s of simulation time, independent of the initial folded or unfolded conformational ensemble. Our results provide practical guidelines for selecting sampling protocols suitable for quantitative RNA benchmarks and lay the foundation for systematic validation and future refinement of RNA FFs.

biophysics↗

bioRxiv2024

Can We Ever Develop an Ideal RNA Force Field? Lessons Learned from Simulations of UUCG RNA Tetraloop and Other Systems.

Molecular dynamics (MD) simulations are an important and well-established tool for investigating RNA structural dynamics, but their accuracy relies heavily on the quality of the employed force field (ff). In this work, we present a comprehensive evaluation of widely used pair-additive and polarizable RNA ffs using the challenging UUCG tetraloop (TL) benchmark system. Extensive standard MD simulations, initiated from the NMR structure of the 14-mer UUCG TL, revealed that most ffs did not maintain the native state, instead favoring alternative loop conformations. Notably, three very recent variants of pair-additive ffs, OL3CP-gHBfix21, DESAMBER, and OL3R2.7, successfully preserved the native structure over a 10 x 20 {micro}s timescale. To further assess these ffs, we performed enhanced sampling folding simulations of the shorter 8-mer UUCG TL, starting from the single-stranded conformation. Estimated folding free energies ({Delta}G{degrees}fold) varied significantly among these three ffs, with values of 0.0 {+/-} 0.6 kcal/mol, 2.4 {+/-} 0.8 kcal/mol, and 7.4 {+/-} 0.2 kcal/mol for OL3CP-gHBfix21, DESAMBER, and OL3R2.7, respectively. The {Delta}G{degrees}fold value from OL3CP-gHBfix21 was closest to experimental data, while the higher {Delta}G{degrees}fold values from DESAMBER and OL3R2.7 were unexpected, suggesting an over- or underestimation of key interactions within the folded and mis(un)folded ensembles. These discrepancies led us to further test DESAMBER and OL3R2.7 ffs on additional RNA and DNA systems, where further performance issues were observed. Our results emphasize the complexity of accurately modeling RNA dynamics and suggest that creating an RNA ff capable of reliably performing across a wide range of RNA motifs remains extremely challenging. In conclusion, our study provides valuable insights into the capabilities of current RNA ffs and highlights key areas for future ff development.

biophysics↗