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

High Resolution Image Download MS PowerPoint Slide 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 ff s 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 ff s did not maintain the native state, instead favoring alternative loop conformations. Notably, three very recent variants of pair-additive ff s, OL3 CP –gHBfix21, DES-Amber, and OL3 R2.7, successfully preserved the native structure over a 10 × 20 μs time scale. To further assess these ff s, we performed enhanced sampling folding simulations of the shorter 8-mer UUCG TL, starting from the single-stranded conformation. Estimated folding free energies (Δ G ° fold ) varied significantly among these three ff s, with values of 0.0 ± 0.6, 2.4 ± 0.8, and 7.4 ± 0.2 kcal/mol for OL3 CP –gHBfix21, DES-Amber, and OL3 R2.7, respectively. The Δ G ° fold value predicted by the OL3 CP –gHBfix21 ff was closest to experimental estimates, ranging from −1.6 to −0.7 kcal/mol. In contrast, the higher Δ G ° fold values obtained using DES-Amber and OL3 R2.7 were unexpected, suggesting that key interactions are inaccurately described in the folded, unfolded, or misfolded ensembles. These discrepancies led us to further test DES-Amber and OL3 R2.7 ff s 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 systems remains extremely challenging. In conclusion, our study provides valuable insights into the capabilities of current RNA ff s and highlights key areas for future ff development.