Can We Ever Developan Ideal RNA Force Field? LessonsLearned from Simulations of the 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 (<i>ff</i>). In this work, we present a comprehensive evaluation of widely used pair-additive and polarizable RNA <i>ff</i>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 <i>ff</i>s did not maintain the native state, instead favoring alternative loop conformations. Notably, three very recent variants of pair-additive <i>ff</i>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 <i>ff</i>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<i>G</i>°_fold) varied significantly among these three <i>ff</i>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<i>G</i>°_fold value predicted by the OL3_CP–gHBfix21 <i>ff</i> was closest to experimental estimates, ranging from −1.6 to −0.7 kcal/mol. In contrast, the higher Δ<i>G</i>°_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 <i>ff</i>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 <i>ff</i> 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 <i>ff</i>s and highlights key areas for future <i>ff</i> development.