Molecular dynamics (MD) simulations became a leading tool for investigation of structural dynamics of nucleic acids. Despite recent efforts to improve the empirical potentials (force fields, ffs), RNA ffs have persisting deficiencies, which hamper their utilization in quantitatively accurate simulations. Previous studies have shown that at least two salient problems contribute to difficulties in the description of free-energy landscapes of small RNA motifs: (i) excessive stabilization of the unfolded single-stranded RNA ensemble by intramolecular base–phosphate and sugar–phosphate interactions and (ii) destabilization of the native folded state by underestimation of stability of base pairing. Here, we introduce a general ff term (gHBfix) that can selectively fine-tune nonbonding interaction terms in RNA ffs, in particular, the H bonds. The gHBfix potential affects the pairwise interactions between all possible pairs of the specific atom types, while all other interactions remain intact; i.e., it is not a structure-based model. In order to probe the ability of the gHBfix potential to refine the ff nonbonded terms, we performed an extensive set of folding simulations of RNA tetranucleotides and tetraloops. On the basis of these data, we propose particular gHBfix parameters to modify the AMBER RNA ff. The suggested parametrization significantly improves the agreement between experimental data and the simulation conformational ensembles, although our current ff version still remains far from being flawless. While attempts to tune the RNA ffs by conventional reparametrizations of dihedral potentials or nonbonded terms can lead to major undesired side effects, as we demonstrate for some recently published ffs, gHBfix has a clear promising potential to improve the ff performance while avoiding introduction of major new imbalances.
The hairpin ribozyme is a prominent member of the group of small catalytic RNAs (RNA enzymes or ribozymes) because it does not require metal ions to achieve catalysis. Biochemical and structural data have implicated guanine 8 (G8) and adenine 38 (A38) as catalytic participants in cleavage and ligation catalyzed by the hairpin ribozyme, yet their exact role in catalysis remains disputed. To gain insight into dynamics in the active site of a minimal self-cleaving hairpin ribozyme, we have performed extensive classical, explicit-solvent molecular dynamics (MD) simulations on timescales of 50-150 ns. Starting from the available X-ray crystal structures, we investigated the structural impact of the protonation states of G8 and A38, and the inactivating A−1(2′-methoxy) substitution employed in crystallography. Our simulations reveal that a canonical G8 agrees well with the crystal structures while a deprotonated G8 profoundly distorts the active site. Thus MD simulations do not support a straightforward participation of the deprotonated G8 in catalysis. By comparison, the G8 enol tautomer is structurally well tolerated, causing only local rearrangements in the active site. Furthermore, a protonated A38H + is more consistent with the crystallography data than a canonical A38. The simulations thus support the notion that A38H + is the dominant form in the crystals, grown at pH 6. In most simulations, the canonical A38 departs from the scissile phosphate and substantially perturbs the structures of active site and S-turn. Yet, we occasionally also observe formation of a stable A−1(2′-OH)…A38(N1) hydrogen bond, which documents the ability of the ribozyme to form this hydrogen bond, consistent with a potential role of A38 as general base catalyst. The presence of this hydrogen bond is, however, incompatible with the expected in-line attack angle necessary for self-cleavage, requiring a rapid transition of the deprotonated 2′-oxyanion to a position more favorable for in-line attack after proton transfer from A−1(2′-OH) to A38(N1). The simulations revealed a potential force field artifact, occasional but irreversible formation of 'ladder-like', underwound A-RNA structure in one of the external helices. Although it does not affect the catalytic center of the hairpin ribozyme, further studies are under way to better assess possible influence of such force field behavior on long RNA simulations.
Explicit solvent atomistic molecular dynamics (MD) simulations represent an established technique to study structural dynamics of RNA molecules and an important complement for diverse experimental methods. However, performance of molecular mechanical (MM) force fields (ff's) remains far from satisfactory even after decades of development, as apparent from a problematic structural description of some important RNA motifs. Actually, some of the smallest RNA molecules belong to the most challenging systems for MD simulations and, among them, the UUCG tetraloop is saliently difficult. We report a detailed analysis of UUCG MD simulations, depicting the sequence of events leading to the loss of the UUCG native state during MD simulations. The total amount of MD simulation data analyzed in this work is close to 1.3 ms. We identify molecular interactions, backbone conformations, and substates that are involved in the process. Then, we unravel specific ff deficiencies using diverse quantum mechanical/molecular mechanical (QM/MM) and QM calculations. Comparison between the MM and QM methods shows discrepancies in the description of the 5′-flanking phosphate moiety and both signature sugar−base interactions. Our work indicates that poor behavior of the UUCG tetraloop in simulations is a complex issue that cannot be attributed to one dominant and straightforwardly correctable factor. Instead, there is a concerted effect of multiple ff inaccuracies that are coupled and amplifying each other. We attempted to improve the simulation behavior by some carefully tailored interventions, but the results were still far from satisfactory, underlying the difficulties in development of accurate nucleic acid ff's.
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