Toward Convergence in Folding Simulations of RNA Tetraloops : Comparison of Enhanced Sampling Techniques and Effects of Force Field Modifications br

Atomistic molecular dynamics simulations repre-sent an established technique for investigation of RNA structuraldynamics. Despite continuous development, contemporary RNAsimulations still suffer from suboptimal accuracy of empiricalpotentials (forcefields,ffs) and sampling limitations. Developmentof efficient enhanced sampling techniques is important for tworeasons. First, they allow us to overcome the sampling limitations,and second, they can be used to quantifyffimbalances providedthey reach a sufficient convergence. Here, we study two RNAtetraloops (TLs), namely the GAGA and UUCG motifs. Weperform extensive folding simulations and calculate folding freeenergies (Delta Gfold degrees) with the aim to compare different enhancedsampling techniques and to test several modifications of thenonbonded terms extending the AMBER OL3 RNAff. We demonstrate that replica-exchange solute tempering (REST2)simulations with 12-16 replicas do not show any sign of convergence even when extended to a timescale of 120 mu s per replica.However, the combination of REST2 with well-tempered metadynamics (ST-MetaD) achieves good convergence on a timescale of5-10 mu s per replica, improving the sampling efficiency by at least 2 orders of magnitude. Effects offfmodifications on Delta Gfold degrees energies were initially explored by the reweighting approach and then validated by new simulations. We tested several manuallyprepared variants of the gHBfix potential which improve stability of the native state of both TLs by similar to 2 kcal/mol. This is sufficient toconveniently stabilize the folded GAGA TL while the UUCG TL still remains under-stabilized. Appropriate adjustment of van derWaals parameters for C-HmiddotmiddotmiddotO5 ' base-phosphate interaction may further stabilize the native states of both TLs by similar to 0.6 kcal/mol