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

Abstract

Atomistic molecular dynamics simulations represent an 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 us 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 a timescale of 120 μs per replica. However, the combination of REST2 with well-tempered metadynamics (ST-MetaD) achieves good convergence on a timescale of 5−10 μs per replica, improving the sampling efficiency by at least 2 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 the gHBfix potential which improve stability of the native state of both TLs by ∼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 may further stabilize the native states of both TLs by ∼0.6 kcal/mol.

Automatic Learning of Hydrogen-Bond Fixes in the AMBER RNA Force Field

“… 8 In a separate manuscript, we showed that using ST-MetaD with MetaD on eRMSD greatly improved the performance of pure ST for RNA tetraloops. 51 Similar conclusions were drawn in ref ( 54 ), where parallel tempering-MetaD 55 , 56 with MetaD on the number of native contacts, 57 a variable highly correlated with eRMSD, was suggested to be significantly more efficient than pure parallel tempering for a GNRA tetraloop. ST-MetaD simulations were carried out using a GPU-capable version of GROMACS2018 44 in combination with PLUMED 2.5 58 , 59 (see section 2.7 for more details about implementation of the gHBfix function within PLUMED code and Table 1 in the Supporting Information for a full list of standard as well as enhanced sampling simulations).…”

Lone Pair…π Contacts and Structure Signatures of r(UNCG) Tetraloops, Z-Turns, and Z-Steps: A WebFR3D Survey

“…Recently, several of these contacts were monitored in MD simulations including a labile 0BPh contact involving the G of the first stem base pair [ 54 , 59 ]. Such C-H…O contact [ 88 ] are very subtle and difficult to model although recent parameterization efforts involving a modification of vdW radii improved the sampling of the loops.…”

“…Such C-H…O contact [ 88 ] are very subtle and difficult to model although recent parameterization efforts involving a modification of vdW radii improved the sampling of the loops. Unfortunately, the conclusions of these studies reveal that we are currently far from mastering all interactions that are essential for modeling the apparently “simple” tetraloop systems [ 54 , 59 ].…”

“…A recent report summarized some of these challenges for RNA tetraloops that could not all be resolved by recent parameterization efforts [ 54 , 55 , 56 , 57 , 58 , 59 ]. The characteristic r(UUCG) native structure is lost in ns to μs standard MD simulations or does not fold correctly because of at least two different effects.…”

“…Recently, several of these contacts were monitored in MD simulations including a labile 0BPh contact involving the G of the first stem base pair [54,59]. Such C-H .…”