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, DES-Amber, and OL3R2.7, successfully preserved the native structure over a 10 × 20 μs time scale. 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 (ΔG°fold) varied significantly among these three ffs, with values of 0.0 ± 0.6, 2.4 ± 0.8, and 7.4 ± 0.2 kcal/mol for OL3CP-gHBfix21, DES-Amber, and OL3R2.7, respectively. The ΔG°fold value predicted by the OL3CP-gHBfix21 ff was closest to experimental estimates, ranging from -1.6 to -0.7 kcal/mol. In contrast, the higher ΔG°fold values obtained using DES-Amber and OL3R2.7 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 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 systems 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.

• MeSH
• Nucleic Acid Conformation MeSH
• RNA * chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA * MeSH

Guanine quadruplexes (GQs) play crucial roles in various biological processes, and understanding their folding pathways provides insight into their stability, dynamics, and functions. This knowledge aids in designing therapeutic strategies, as GQs are potential targets for anticancer drugs and other therapeutics. Although experimental and theoretical techniques have provided valuable insights into different stages of the GQ folding, the structural complexity of GQs poses significant challenges, and our understanding remains incomplete. This study introduces a novel computational protocol for folding an entire GQ from single-strand conformation to its native state. By combining two complementary enhanced sampling techniques, we were able to model folding pathways, encompassing a diverse range of intermediates. Although our investigation of the GQ free energy surface (FES) is focused solely on the folding of the all-anti parallel GQ topology, this protocol has the potential to be adapted for the folding of systems with more complex folding landscapes.

• Keywords
• DNA quadruplex, computational folding, enhanced sampling, kinetic partitioning mechanism, metadynamics, molecular dynamics, nudged elastic band, pathCV, transition path sampling,
• MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Nucleic Acid Conformation MeSH
• Molecular Dynamics Simulation MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH

Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy─steered molecular dynamics (SMD) simulations─in combination with standard molecular dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. Using a special set of structural parameters, we found that the vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of the vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage, and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event, and it usually started at the 3'-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.

• MeSH
• Biomechanical Phenomena MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Mechanical Phenomena MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH