Molecular Dynamics Simulations Reveal an Interplay between SHAPE Reagent Binding and RNA Flexibility

The function of RNA molecules usually depends on their overall fold and on the presence of specific structural motifs. Chemical probing methods are routinely used in combination with nearest-neighbor models to determine RNA secondary structure. Among the available methods, SHAPE is relevant due to its capability to probe all RNA nucleotides and the possibility to be used in vivo. However, the structural determinants for SHAPE reactivity and its mechanism of reaction are still unclear. Here molecular dynamics simulations and enhanced sampling techniques are used to predict the accessibility of nucleotide analogs and larger RNA structural motifs to SHAPE reagents. We show that local RNA reconformations are crucial in allowing reagents to reach the 2'-OH group of a particular nucleotide and that sugar pucker is a major structural factor influencing SHAPE reactivity

Atomistic Picture of Opening–Closing Dynamics of DNA Holliday Junction Obtained by Molecular Simulations

Holliday junction (HJ) is a noncanonical four-way DNA structure with a prominent role in DNA repair, recombination, and DNA nanotechnology. By rearranging its four arms, HJ can adopt either closed or open state. With enzymes typically recognizing only a single state, acquiring detailed knowledge of the rearrangement process is an important step toward fully understanding the biological function of HJs. Here, we carried out standard all-atom molecular dynamics (MD) simulations of the spontaneous opening-closing transitions, which revealed complex conformational transitions of HJs with an involvement of previously unconsidered “half-closed” intermediates. Detailed free-energy landscapes of the transitions were obtained by sophisticated enhanced sampling simulations. Because the force field overstabilizes the closed conformation of HJs, we developed a system-specific modification which for the first time allows the observation of spontaneous opening-closing HJ transitions in unbiased MD simulations and opens the possibilities for more accurate HJ computational studies of biological processes and nanomaterials

Sensitivity of the RNA structure to ion conditions as probed by molecular dynamics simulations of common canonical RNA duplexes

RNA molecules play a key role in countless biochemical processes. RNA interactions, which are of highly diverse nature, are determined by the fact that RNA is a highly negatively charged polyelectrolyte, which leads to intimate interactions with an ion atmosphere. Although RNA molecules are formally single stranded, canonical (Watson−Crick) duplexes are key components of folded RNAs. A double-stranded (ds) RNA is also important for the design of RNA-based nanostructures and assemblies. Despite the fact that the description of canonical dsRNA is considered the least problematic part of RNA modeling, the imperfect shape and flexibility of dsRNA can lead to imbalances in the simulations of larger RNAs and RNA-containing assemblies. We present a comprehensive set of molecular dynamics (MD) simulations of four canonical A-RNA duplexes. Our focus was directed toward the characterization of the influence of varying ion concentrations and of the size of the solvation box. We compared several water models and four RNA force fields. The simulations showed that the A-RNA shape was most sensitive to the RNA force field, with some force fields leading to a reduced inclination of the A-RNA duplexes. The ions and water models played a minor role. The effect of the box size was negligible, and even boxes with a small fraction of the bulk solvent outside the RNA hydration sphere were sufficient for the simulation of the dsRNA.Web of Science6372146213

D8.1 : Data Management Plan

The Data Management Plan lays out our planning for handling main aspects of the life cycle of the project data (data organisation and long-term storage, access, preservation, and sharing). This document also includes a preliminary specification of outputs (what data will be generated during the project). It is a living document and will be continuously updated during the project.Ostrav

Exploring RNA structure and dynamics through enhanced sampling simulations

RNA function is intimately related to its structural dynamics. Molecular dynamics simulations are useful for exploring biomolecular flexibility but are severely limited by the accessible timescale. Enhanced sampling methods allow this timescale to be effectively extended in order to probe biologically relevant conformational changes and chemical reactions. Here, we review the role of enhanced sampling techniques in the study of RNA systems. We discuss the challenges and promises associated with the application of these methods to force-field validation, exploration of conformational landscapes and ion/ligand-RNA interactions, as well as catalytic pathways. Important technical aspects of these methods, such as the choice of the biased collective variables and the analysis of multi-replica simulations, are examined in detail. Finally, a perspective on the role of these methods in the characterization of RNA dynamics is provided

Analyzing and Biasing Simulations with PLUMED

This chapter discusses how the PLUMED plugin for molecular dynamics can be used to analyze and bias molecular dynamics trajectories. The chapter begins by introducing the notion of a collective variable and by then explaining how the free energy can be computed as a function of one or more collective variables. A number of practical issues mostly around periodic boundary conditions that arise when these types of calculations are performed using PLUMED are then discussed. Later parts of the chapter discuss how PLUMED can be used to perform enhanced sampling simulations that introduce simulation biases or multiple replicas of the system and Monte Carlo exchanges between these replicas. This section is then followed by a discussion on how free-energy surfaces and associated error bars can be extracted from such simulations by using weighted histogram and block averaging techniques

Multiscale modelling of phosphate…π contacts in RNA U-turns reveals AMBER force-field deficiencies

Phosphate…π, also called anion…π, contacts occur between nucleobases and phosphate OP oxygens in r(GNRA) and r(UNNN) U-turn motifs (N = A,G,C,U; R = A,G). We investigated these contacts in detail by using state-of-the-art quantum chemical methods (QM) to characterize some of their physico-chemical properties and to evaluate the ability of the AMBER force field (AFF) to describe these contacts. We found that AFF interaction energies of phosphate…π contacts calculated for model dimethyl phosphate…nucleobase systems are less stabilizing in comparison with double-hybrid DFT methods and that the minimum contact distances are stretched for all nucleobase systems. This distance stretch is also observed in large-scale AFF computations on several r(gcGNRAgc) tetraloop hairpins when compared to QM/MM. Further, classical molecular dynamics (MD) simulations of these tetraloop hairpins confirm this distance stretch and reveal shifted OP2/nucleobase positions when compared to experimental data extracted from high-resolution X ray/cryo EM structures (≤ 2.5 Å) of r(GNRA) tetraloops using the WebFR3D bioinformatic tool. We propose that discrepancies between QM and AFF are caused by a combination of missing polarization, too large AFF Lennard-Jones (LJ) radii of nucleobase carbon atoms and exaggerated short-range repulsion due to an approximate r−12 LJ repulsive term. We put these results in regard with those obtained in earlier investigations on lone pair…π contacts occurring in CpG Z-steps. Charge-transfer calculations do not support any significant n->π* donation effects and hence this label is inappropriate. We also investigated thiophosphate…π contacts for which we calculated less stabilizing interaction energies than for the phosphate…π contacts. We thus challenge suggestions that the experimentally observed enhanced thermodynamic stability of phosphorothioated r(GNRA) tetraloops can be straightforwardly explained by larger London dispersion

Toward convergence in folding simulations of RNA tetraloops: Comparison of enhanced sampling techniques and effects of force field modifications

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 (ΔGfold°) 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 ΔGfold° 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.Web of Science1842656264

Automatic learning of hydrogen-bond fixes in an AMBER RNA force field

The capability of current force fields to reproduce RNA structural dynamics is limited. Several methods have been developed to take advantage of experimental data in order to enforce agreement with experiments. We herein extend an existing framework, which allows arbitrarily chosen force-field correction terms to be fitted by quantification of the discrepancy between observables back-calculated from simulation and corresponding experiments. We apply a robust regularization protocol to avoid overfitting, and additionally introduce and compare a number of different regularization strategies, namely L1-, L2-, Kish Size-, Relative Kish Size- and Relative Entropy-penalties. The training set includes a GACC tetramer as well as more challenging systems, namely gcGAGAgc and gcUUCGgc RNA tetraloops. Specific intramolecular hydrogen bonds in the AMBER RNA force field are corrected with automatically determined parameters that we call gHBfix$_{opt}$. A validation involving a separate simulation of a system present in the training set (gcUUCGgc) and new systems not seen during training (CAAU and UUUU tetramers) displays improvements regarding native population of the tetraloop as well as good agreement with NMR-experiments for tetramers when using the new parameters. Then we simulate folded RNAs (a kink-turn and L1 stalk rRNA) including hydrogen bond types not sufficiently present in the training set. This allows a final modification of the parameter set which is named gHBfix21 and is suggested to be applicable to a wider range of RNA systems.Comment: Supporting information included in ancillary file