Towards Convergence in Folding Simulations of RNA Tetraloops: Comparison of Enhanced Sampling Techniques and Effects of Force Field Corrections

ABSTRACT Atomistic molecular dynamics (MD) simulations represent established technique for investigation of RNA structural dynamics. Despite continuous development, contemporary RNA simulations still suffer from suboptimal accuracy of empirical potentials (force fields, ffs ) and sampling limitations. Development of efficient enhanced sampling techniques is important for two reasons. First, they allow to overcome the sampling limitations and, second, they can be used to quantify ff imbalances provided they reach a sufficient convergence. Here, we study two RNA tetraloops (TLs), namely the GAGA and UUCG motifs. We perform extensive folding simulations and calculate folding free energies (ΔG fold ) with the aim to compare different enhanced sampling techniques and to test several modifications of the nonbonded terms extending the AMBER OL3 RNA ff . We demonstrate that replica exchange solute tempering (REST2) simulations with 12-16 replicas do not show any sign of convergence even when extended to time scale of 120 μs per replica. However, combination of REST2 with well-tempered metadynamics (ST-MetaD) achieves good convergence on a time-scale of 5-10 μs per replica, improving the sampling efficiency by at least two orders of magnitude. Effects of ff modifications on ΔG fold energies were initially explored by the reweighting approach and then validated by new simulations. We tested several manually-prepared variants of gHBfix potential which improve stability of the native state of both TLs by up to ~2 kcal/mol. This is sufficient to conveniently stabilize the folded GAGA TL while the UUCG TL still remains under-stabilized. Appropriate adjustment of van der Waals parameters for C-H…O5’ base-phosphate interaction are also shown to be capable of further stabilizing the native states of both TLs by ~0.6 kcal/mol.