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
• Nucleic Acid Conformation MeSH
• RNA * chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA * MeSH

Lipid-mediated delivery of active pharmaceutical ingredients (API) opened new possibilities in advanced therapies. By encapsulating an API into a lipid nanocarrier (LNC), one can safely deliver APIs not soluble in water, those with otherwise strong adverse effects, or very fragile ones such as nucleic acids. However, for the rational design of LNCs, a detailed understanding of the composition-structure-function relationships is missing. This review presents currently available computational methods for LNC investigation, screening, and design. The state-of-the-art physics-based approaches are described, with the focus on molecular dynamics simulations in all-atom and coarse-grained resolution. Their strengths and weaknesses are discussed, highlighting the aspects necessary for obtaining reliable results in the simulations. Furthermore, a machine learning, i.e., data-based learning, approach to the design of lipid-mediated API delivery is introduced. The data produced by the experimental and theoretical approaches provide valuable insights. Processing these data can help optimize the design of LNCs for better performance. In the final section of this Review, state-of-the-art of computer simulations of LNCs are reviewed, specifically addressing the compatibility of experimental and computational insights.

• Keywords
• ionizable lipid, lipid nanocarrier, lipid nanoparticle, liposome, molecular simulation, vesicle,
• MeSH
• Pharmaceutical Preparations chemistry   administration & dosage   MeSH
• Drug Delivery Systems * methods   MeSH
• Humans MeSH
• Lipids * chemistry   MeSH
• Nanoparticles chemistry   MeSH
• Drug Carriers * chemistry   MeSH
• Computer Simulation MeSH
• Molecular Dynamics Simulation MeSH
• Machine Learning MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Pharmaceutical Preparations MeSH
• Lipids * MeSH
• Drug Carriers * MeSH

Glycans, consisting of covalently linked sugar units, are a major class of biopolymers essential to all known living organisms. To better understand their biological functions and further applications in fields from biomedicine to materials science, detailed knowledge of their structure is essential. However, due to the extraordinary complexity and conformational flexibility of glycans, state-of-the-art glycan analysis methods often fail to provide structural information with atomic precision. Here, we combine electrospray deposition in ultra-high vacuum with non-contact atomic force microscopy and theoretical calculations to unravel the structure of β-cyclodextrin, a cyclic glucose oligomer, with atomic-scale detail. Our results, established on the single-molecule level, reveal the different adsorption geometries and conformations of β-cyclodextrin. The position of individual hydroxy groups and the location of the stabilizing intramolecular H-bonds are deduced from atomically resolved images, enabling the unambiguous assignment of the molecular structure and demonstrating the potential of the method for glycan analysis.

• Publication type
• Journal Article MeSH

Mixed double helices formed by RNA and DNA strands, commonly referred to as hybrid duplexes or hybrids, are essential in biological processes like transcription and reverse transcription. They are also important for their applications in CRISPR gene editing and nanotechnology. Yet, despite their significance, the hybrid duplexes have been seldom modeled by atomistic molecular dynamics methodology, and there is no benchmark study systematically assessing the force-field performance. Here, we present an extensive benchmark study of polypurine tract (PPT) and Dickerson-Drew dodecamer hybrid duplexes using contemporary and commonly utilized pairwise additive and polarizable nucleic acid force fields. Our findings indicate that none of the available force-field choices accurately reproduces all the characteristic structural details of the hybrid duplexes. The AMBER force fields are unable to populate the C3'-endo (north) pucker of the DNA strand and underestimate inclination. The CHARMM force field accurately describes the C3'-endo pucker and inclination but shows base pair instability. The polarizable force fields struggle with accurately reproducing the helical parameters. Some force-field combinations even demonstrate a discernible conflict between the RNA and DNA parameters. In this work, we offer a candid assessment of the force-field performance for mixed DNA/RNA duplexes. We provide guidance on selecting utilizable force-field combinations and also highlight potential pitfalls and best practices for obtaining optimal performance.

• MeSH
• DNA * chemistry   MeSH
• Nucleic Acid Conformation * MeSH
• Base Pairing MeSH
• RNA * chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA * MeSH
• RNA * 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
• Phosphates * MeSH
• Molecular Conformation MeSH
• Nucleotide Motifs MeSH
• RNA * chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Phosphates * MeSH
• RNA * MeSH

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.

• MeSH
• Ions chemistry   MeSH
• Nucleic Acid Conformation MeSH
• RNA * chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ions MeSH
• RNA * MeSH
• Water 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
• ELAV-Like Protein 1 metabolism   MeSH
• RNA Recognition Motif genetics   MeSH
• RNA-Binding Proteins * metabolism   MeSH
• RNA * chemistry   MeSH
• RNA Recognition Motif Proteins metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ELAV-Like Protein 1 MeSH
• RNA-Binding Proteins * MeSH
• RNA * MeSH
• RNA Recognition Motif Proteins 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, Ribosomal MeSH
• RNA * chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Hydrogen MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA, Ribosomal MeSH
• RNA * MeSH
• Hydrogen MeSH

The conserved protein Hfq is a key factor in the RNA-mediated control of gene expression in most known bacteria. The transient intermediates Hfq forms with RNA support intricate and robust regulatory networks. In Pseudomonas, Hfq recognizes repeats of adenine-purine-any nucleotide (ARN) in target mRNAs via its distal binding side, and together with the catabolite repression control (Crc) protein, assembles into a translation-repression complex. Earlier experiments yielded static, ensemble-averaged structures of the complex, but details of its interface dynamics and assembly pathway remained elusive. Using explicit solvent atomistic molecular dynamics simulations, we modeled the extensive dynamics of the Hfq-RNA interface and found implications for the assembly of the complex. We predict that syn/anti flips of the adenine nucleotides in each ARN repeat contribute to a dynamic recognition mechanism between the Hfq distal side and mRNA targets. We identify a previously unknown binding pocket that can accept any nucleotide and propose that it may serve as a 'status quo' staging point, providing nonspecific binding affinity, until Crc engages the Hfq-RNA binary complex. The dynamical components of the Hfq-RNA recognition can speed up screening of the pool of the surrounding RNAs, participate in rapid accommodation of the RNA on the protein surface, and facilitate competition among different RNAs. The register of Crc in the ternary assembly could be defined by the recognition of a guanine-specific base-phosphate interaction between the first and last ARN repeats of the bound RNA. This dynamic substrate recognition provides structural rationale for the stepwise assembly of multicomponent ribonucleoprotein complexes nucleated by Hfq-RNA binding.

• Keywords
• ARN repeats, Crc protein, Hfq protein, RNA metabolism, RNA-binding protein, dynamic recognition, molecular dynamics, protein–nucleic acid interaction,
• MeSH
• RNA, Bacterial chemistry   genetics   metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Protein Conformation MeSH
• Nucleotide Motifs * MeSH
• Host Factor 1 Protein chemistry   genetics   metabolism   MeSH
• Pseudomonas aeruginosa genetics   metabolism   MeSH
• Gene Expression Regulation, Bacterial * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Bacterial MeSH
• Host Factor 1 Protein MeSH

We recently showed that Saccharomyces cerevisiae telomeric DNA can fold into an unprecedented pseudocircular G-hairpin (PGH) structure. However, the formation of PGHs in the context of extended sequences, which is a prerequisite for their function in vivo and their applications in biotechnology, has not been elucidated. Here, we show that despite its 'circular' nature, PGHs tolerate single-stranded (ss) protrusions. High-resolution NMR structure of a novel member of PGH family reveals the atomistic details on a junction between ssDNA and PGH unit. Identification of new sequences capable of folding into one of the two forms of PGH helped in defining minimal sequence requirements for their formation. Our time-resolved NMR data indicate a possibility that PGHs fold via a complex kinetic partitioning mechanism and suggests the existence of K+ ion-dependent PGH folding intermediates. The data not only provide an explanation of cation-type-dependent formation of PGHs, but also explain the unusually large hysteresis between PGH melting and annealing noted in our previous study. Our findings have important implications for DNA biology and nanotechnology. Overrepresentation of sequences able to form PGHs in the evolutionary-conserved regions of the human genome implies their functionally important biological role(s).

• MeSH
• Nucleic Acid Conformation MeSH
• DNA, Circular chemistry   MeSH
• Models, Molecular MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Nucleotide Motifs MeSH
• Base Pairing MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Stereoisomerism MeSH
• Telomere chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Circular MeSH