Molecular dynamics (MD) simulations are an important and well-established tool for investigating RNA structural dynamics, but their accuracy relies heavily on the quality of the employed force field (ff). In this work, we present a comprehensive evaluation of widely used pair-additive and polarizable RNA ffs using the challenging UUCG tetraloop (TL) benchmark system. Extensive standard MD simulations, initiated from the NMR structure of the 14-mer UUCG TL, revealed that most ffs did not maintain the native state, instead favoring alternative loop conformations. Notably, three very recent variants of pair-additive ffs, OL3CP-gHBfix21, DES-Amber, and OL3R2.7, successfully preserved the native structure over a 10 × 20 μs time scale. To further assess these ffs, we performed enhanced sampling folding simulations of the shorter 8-mer UUCG TL, starting from the single-stranded conformation. Estimated folding free energies (ΔG°fold) varied significantly among these three ffs, with values of 0.0 ± 0.6, 2.4 ± 0.8, and 7.4 ± 0.2 kcal/mol for OL3CP-gHBfix21, DES-Amber, and OL3R2.7, respectively. The ΔG°fold value predicted by the OL3CP-gHBfix21 ff was closest to experimental estimates, ranging from -1.6 to -0.7 kcal/mol. In contrast, the higher ΔG°fold values obtained using DES-Amber and OL3R2.7 were unexpected, suggesting that key interactions are inaccurately described in the folded, unfolded, or misfolded ensembles. These discrepancies led us to further test DES-Amber and OL3R2.7 ffs on additional RNA and DNA systems, where further performance issues were observed. Our results emphasize the complexity of accurately modeling RNA dynamics and suggest that creating an RNA ff capable of reliably performing across a wide range of RNA systems remains extremely challenging. In conclusion, our study provides valuable insights into the capabilities of current RNA ffs and highlights key areas for future ff development.

• MeSH
• konformace nukleové kyseliny MeSH
• RNA * chemie   MeSH
• simulace molekulární dynamiky * MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA * MeSH

The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

• MeSH
• 3' nepřekládaná oblast * MeSH
• chybné párování bází * MeSH
• COVID-19 virologie   MeSH
• genom virový MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• nukleotidové motivy * MeSH
• párování bází MeSH
• Plasmodium falciparum genetika   MeSH
• RNA virová * chemie   genetika   MeSH
• SARS-CoV-2 * genetika   chemie   MeSH
• simulace molekulární dynamiky MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast * MeSH
• RNA virová * MeSH

Molecular dynamics (MD) simulations represent an established tool to study RNA molecules. The outcome of MD studies depends, however, on the quality of the force field (ff). Here we suggest a correction for the widely used AMBER OL3 ff by adding a simple adjustment of the nonbonded parameters. The reparameterization of the Lennard-Jones potential for the -H8···O5'- and -H6···O5'- atom pairs addresses an intranucleotide steric clash occurring in the type 0 base-phosphate interaction (0BPh). The nonbonded fix (NBfix) modification of 0BPh interactions (NBfix0BPh modification) was tuned via a reweighting approach and subsequently tested using an extensive set of standard and enhanced sampling simulations of both unstructured and folded RNA motifs. The modification corrects minor but visible intranucleotide clash for the anti nucleobase conformation. We observed that structural ensembles of small RNA benchmark motifs simulated with the NBfix0BPh modification provide better agreement with experiments. No side effects of the modification were observed in standard simulations of larger structured RNA motifs. We suggest that the combination of OL3 RNA ff and NBfix0BPh modification is a viable option to improve RNA MD simulations.

• MeSH
• fosfáty * MeSH
• molekulární konformace MeSH
• nukleotidové motivy MeSH
• RNA * chemie   MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfáty * MeSH
• RNA * MeSH

Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs.

• Klíčová slova
• MD simulations, NMR, RNA structure, U-turn, U:U base pair, pseudouridine,
• MeSH
• konformace nukleové kyseliny MeSH
• párování bází MeSH
• pseudouridin * genetika   MeSH
• RNA * genetika   chemie   MeSH
• uridin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pseudouridin * MeSH
• RNA * MeSH
• uridin MeSH

Recognition of single-stranded RNA (ssRNA) by RNA recognition motif (RRM) domains is an important class of protein-RNA interactions. Many such complexes were characterized using nuclear magnetic resonance (NMR) and/or X-ray crystallography techniques, revealing ensemble-averaged pictures of the bound states. However, it is becoming widely accepted that better understanding of protein-RNA interactions would be obtained from ensemble descriptions. Indeed, earlier molecular dynamics simulations of bound states indicated visible dynamics at the RNA-RRM interfaces. Here, we report the first atomistic simulation study of spontaneous binding of short RNA sequences to RRM domains of HuR and SRSF1 proteins. Using a millisecond-scale aggregate ensemble of unbiased simulations, we were able to observe a few dozen binding events. HuR RRM3 utilizes a pre-binding state to navigate the RNA sequence to its partially disordered bound state and then to dynamically scan its different binding registers. SRSF1 RRM2 binding is more straightforward but still multiple-pathway. The present study necessitated development of a goal-specific force field modification, scaling down the intramolecular van der Waals interactions of the RNA which also improves description of the RNA-RRM bound state. Our study opens up a new avenue for large-scale atomistic investigations of binding landscapes of protein-RNA complexes, and future perspectives of such research are discussed.

• MeSH
• HuR protein metabolismus   MeSH
• motiv rozpoznávající RNA genetika   MeSH
• proteiny vázající RNA * metabolismus   MeSH
• RNA * chemie   MeSH
• RRM proteiny metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HuR protein MeSH
• proteiny vázající RNA * MeSH
• RNA * MeSH
• RRM proteiny MeSH

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. Here, we 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 gHBfixopt. 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 the 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.

• MeSH
• RNA ribozomální MeSH
• RNA * chemie   MeSH
• simulace molekulární dynamiky * MeSH
• vodík MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA ribozomální MeSH
• RNA * MeSH
• vodík MeSH