Biology subjects

Sponer, J.

Publications and source records attributed to Sponer, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Parallel DNA Holliday Junctions: Myth or Reality? An Atomistic Molecular Dynamics Study

Holliday junctions (HJs) are key intermediates of homologous recombination and fundamental building blocks in DNA nanotechnology. Although the canonical antiparallel stacked-X conformation has been extensively characterized by X-ray crystallography, whether parallel HJ conformations exist in aqueous solution remains unresolved. Here, we address this question using extensive atomistic molecular dynamics (MD) simulations and replica-exchange umbrella sampling (REUS) free-energy calculations. Starting from canonical antiparallel HJs, standard MD simulations occasionally revealed spontaneous transitions to parallel conformations on the microsecond timescale without disrupting the DNA duplexes or passing through an open junction intermediate. REUS free-energy profiles confirmed the antiparallel state as the global minimum but also identified the parallel conformation as a well-defined local minimum, indicating it is thermodynamically metastable despite an estimated solution population below 1%. The combination of low equilibrium occupancy, microsecond interconversion dynamics, and the surprisingly close structural similarity between antiparallel and parallel junctions provides a plausible explanation for the lack of direct experimental detection. We further found that the free-energy landscape is only weakly affected by branching-point sequence and salt concentration. These results reconcile the apparent absence of experimental evidence for parallel HJs with their structural feasibility in solution and offer a fresh perspective on the historical debate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/737670v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1616b7forg.highwire.dtl.DTLVardef@d868c9org.highwire.dtl.DTLVardef@1c795cborg.highwire.dtl.DTLVardef@3b52b9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Enhanced RNA Formation Under Amine-Rich Local Atmospheres from 2',3'- Cyclic Nucleotides

The core biopolymers (DNA, RNA and proteins) are assembled from their monomers under conditions that avoid water. RNA is crucial for the Origin of Life. When cleaved from its polymerized state, RNA first transitions to nucleoside 2,3-cyclic phosphates. In the reverse direction, RNA polymerizes from 2,3-cyclic monomers in dry states, creating short oligomers that then can ligate on a template under aqueous, alkaline conditions. We studied the role of the counterions in polymerization of 2,3-cyclic nucleotides under geologically plausible settings. Through experiments and simulations, we demonstrate that the presence of ammonium and alkylammonium counterions greatly improves RNA polymerization. The otherwise less reactive cytidine containing monomers formed polyC sequences of up to heptamers; copolymers of AU, GC, or GCAU were detected up to hexamers. Our findings suggest three reasons for this: (1) (Alkyl)ammonium cations form hydrogen bonds with phosphates, (2) their alkaline pKa value can trigger general base catalysis, and (3) (alkyl)ammonium salts naturally form dry, anhydrous materials. The findings indicate that pyrolyzed organic tars creating ammonia-rich gas pockets in subsurface rocks could have enhanced the early evolution of RNA. TOC image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/713775v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@1adc431org.highwire.dtl.DTLVardef@12b8da0org.highwire.dtl.DTLVardef@5f187dorg.highwire.dtl.DTLVardef@140ed1a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

From gHBfix to NBfix: Reweighting-Driven Refinement of Hydrogen-Bond Interactions in RNA Force Fields

Understanding RNA structural dynamics is essential for elucidating its biological functions, and molecular dynamics (MD) simulations provide an important atomistic complement to experimental approaches. However, the predictive power of MD is fundamentally limited by the accuracy of the underlying empirical Force Fields (FFs), particularly in capturing the delicate balance of non-bonded interactions. Here, we present a systematic reparameterization strategy that replaces the external gHBfix19 hydrogen-bond (H-bond) correction potential with an equivalent set of NBfix Lennard-Jones modifications within a state-of-the-art RNA FF. Using a quantitatively converged temperature replica-exchange MD ensemble of the GAGA tetraloop, we employed a reweighting-based optimization protocol to derive NBfix parameters that reproduce the thermodynamic effects of the original gHBfix19 terms. Sequential optimization of individual gHBfix19 components proved essential to ensure stable and transferable parameter refinement. The resulting fully reformulated NBfix-based variant, termed OL3CP-NBfix19, was validated on a representative set of RNA motifs, including tetranucleotides, A-form duplexes, and tetraloops. Across all tested systems, its performance is comparable to that of the reference gHBfix19 FF. By embedding the H-bond corrections directly into the standard non-bonded framework, the NBfix formulation eliminates external biasing potentials, simplifies practical deployment, and reduces computational overhead. Beyond this specific reparameterization, our results demonstrate a practical workflow for translating targeted H-bond corrections into native FF terms for efficient biomolecular simulations.

biophysics↗

bioRxiv2026

Toward Accurate RNA Folding Thermodynamics: Evaluation of Enhanced Sampling Methods for Force Field Benchmarking

Biologically functional RNAs operate near marginal stability, and their rugged free-energy landscapes and profound structural dynamics - typically not captured by structural biology experiments - play decisive roles. Atomistic molecular dynamics (MD) simulations provide a unique means to characterize these features. However, the applicability of atomistic MD is currently limited by accessible simulation timescales and, most importantly, by force-field (FF) accuracy. Folding free energies ({Delta}G{degrees}fold) of small RNA motifs represent well-defined targets for quantitative benchmarking of RNA FFs. In practice, however, obtaining thermodynamic estimates that are sufficiently robust for direct comparison with experimental data remains highly challenging, even for small RNA systems, and many published studies rely on sampling that is not fully converged. Here, we systematically assess the performance of widely used advanced enhanced-sampling techniques using the 8-mer r(gcGAGAgc) tetraloop as a representative benchmark system. We test temperature replica exchange (T-REMD), two solute-tempering variants of replica exchange (REST2 and REHT), as well as well-tempered metadynamics and on-the-fly probability enhanced sampling combined with solute tempering (ST-MetaD and ST-OPES). Among the tested approaches, T-REMD proves to be the most robust, yielding reproducible folding equilibria and consistent estimates of {Delta}G{degrees}fold after approximately 20 s of simulation time, independent of the initial folded or unfolded conformational ensemble. Our results provide practical guidelines for selecting sampling protocols suitable for quantitative RNA benchmarks and lay the foundation for systematic validation and future refinement of RNA FFs.

biophysics↗

bioRxiv2025

Destabilization of Structured RNAs by OPC and TIP4PD Water Models

The four-point OPC water model has recently gained reputation as the preferred choice for molecular dynamics (MD) simulations of nucleic acids and even proteins, providing more realistic reproduction of bulk physical properties of water than the older three-point models. It has been shown to improve for example simulations of unstructured biomolecules such as RNA tetranucleotides or intrinsically disordered proteins. However, the performance for folded RNA structures was not specifically explored. In this study, we present extensive testing of the OPC water model on three different RNAs with highly intricate tertiary structures - the ribosomal L1 stalk RNA-protein protuberance, the mini tetraloop-tetraloop receptor (miniTTR-6) folded RNA, and the GAAA tetraloop-tetraloop receptor homodimer. The OPC performance is directly compared against SPC/E, TIP3P, OPC3, and TIP4P-EW water models along with the common OL3 AMBER RNA force field (FF). We found substantial effect of the water model on simulation behavior. For all three systems, we observe large-scale unfolding of the RNA, and even loss of the L1 stalk protein-RNA interface, when simulated with the OPC. In contrast, the simulations are entirely stable with the three-point water models. The underlying cause seems to be the higher affinity of the OPC waters to H-bond donor and acceptor groups of the RNA, disrupting the native solute-solute interactions. An identical issue can be observed also for the similar and widely used TIP4PD water model combined with the DES-Amber RNA FF. Our findings suggest that caution is warranted when using the four-point OPC and TIP4PD water models for simulations of structured RNAs. In combination with the current AMBER RNA FFs, the three-point water models may provide more realistic alternatives.

biophysics↗

bioRxiv2025

The Kink-Turn Motif: A Powerful Test for Revealing Weaknesses in RNA Force Fields

The kink-turn is a recurrent RNA structural motif that induces a sharp bend (kink) in the A-form RNA helix. It is defined by key structural features, including consecutive sheared AG base pairs, an A-minor interaction, and multiple base-sugar interactions. Accurate representation of these densely packed non-canonical motifs in molecular dynamics simulations poses a significant challenge for contemporary force fields (FFs). Here, we present extended simulations of ribosomal kink-turn 7 (Kt-7) using a broad spectrum of pair-additive and polarizable RNA FFs. None of the tested FFs manage to flawlessly describe all the specific structural features of the Kt-7, which are described in detail in this work. Still, several FFs provide rather acceptable results and should not cause problems in simulations of larger RNAs containing a kink-turn. On aggregate, the widely used OL3 (ff99bsc0{chi}OL3) and polarizable AMOEBA FFs achieve the best performance. On the other hand, some more recently parametrized FF variants struggle to describe the Kt-7s tertiary A-minor interaction - an ubiquitous tertiary contact in RNA. This raises some concerns about the broader applicability of these FFs and suggests that they may be overfitted to small RNA model systems, such as RNA tetranucleotides. The difficulties manifest as either reversible local disruptions of the A-minor interaction and other tertiary contacts or, in some cases, irreversible unkinking of the entire motif. Based on our findings, we strongly suggest including the kink-turn motif in training and benchmarking datasets as the quintessential regression test to enhance the robustness and accuracy of RNA FF parametrization efforts.

biophysics↗

bioRxiv2025

Sequence-Dependent Dynamics of U:U Mismatches in RNA Revealed by Molecular Dynamics Simulations

The uracil:uracil (U:U) base pair is one of the most common mismatches observed in RNA. It is notable for its ability to adopt multiple conformational states depending on its structural environment, particularly on the identity of the flanking canonical base pairs. Here, we employed extensive molecular dynamics (MD) simulations to systematically investigate the conformational dynamics of a U:U mismatch embedded within a model A-form RNA helix, flanked by all possible canonical base pair combinations. We found that the neighboring base pairs strongly influence the preferred conformational states of the U:U mismatch. However, the mismatch still regularly samples the less favored conformations on a timescale of hundreds of nanoseconds. Contrary to previous assumptions, water-mediated conformations are not universally the most stable conformational states for isolated U:U mismatches as some of the variants distinctly prefer the direct H-bonding while others destabilize the U:U mismatch altogether. Our results strongly suggest that the presence of a U:U mismatch introduces local strain into the RNA helix, which can be relieved through dynamic destabilization of either the mismatch itself or the flanking canonical base pairs, occasionally forming a shifting "bubble" of instability. These findings advance our understanding of U:U mismatch behavior in RNA and reveal a complex interplay between local sequence context, structural stability, and RNA dynamics.

biophysics↗

bioRxiv2025

RNA G-Quadruplex Emerges from a Coil-Like Ensemble via Multiple Pathways

RNA G-quadruplexes (rG4s) are vital structural elements in gene regulation and genome stability. In untranslated regions of mRNAs, rG4s influence translation efficiency and mRNA localization. Additionally, rG4s of long non-coding RNAs and telomeric RNA play roles in RNA processing and cellular aging. Despite their significance, atomic-level folding mechanisms of rG4s remain poorly understood due to their complexity. We used enhanced-sampling all-atom molecular dynamics simulations to model the folding of an r(GGGA)3GGG sequence into a parallel-stranded rG4. The folding pathways suggest that RNA initially adopts a compact coil- like ensemble, marked by dynamic guanine stacking and pairing. The three-quartet rG4 builds up gradually from the coil via diverse routes involving strand rearrangements and guanine incorporations. While the folding mechanism is multi-pathway, various two-quartet rG4s seem to be a common transitory ensemble for most routes. Our simulations also exposed force-field imbalances, with the predicted folding free energy of +12.5 kcal/mol deviating from experiments. Additionally, the enhanced sampling protocol, combining well-tempered metadynamics with solute tempering, faced productivity challenges on the multidimensional free-energy surface. Overall, this study provides atomistic insights into rG4 folding, emphasizing compact coil-like ensembles as key precursors, while revealing limitations in simulating non-canonical RNA structures.

biophysics↗

bioRxiv2024

Integrated Database of Force-Field Parameters, Experimental Measurements and Molecular Dynamics Simulations

Molecular Dynamic (MD) simulation is a vital theoretical tool for exploring nucleic acids (RNA, DNA), proteins and other (bio)molecular systems, generating vast amounts of data daily. Efficient storage and possible reuse of this data is a persistent challenge. Here, we introduce IDA (Integrated DAtabase of force fields and datasets from experiments and MD simulations), an innovative database scheme for datasets from various types of MD simulations. IDA supports outputs from different MD approaches, i.e., standard MD simulations, importance sampling techniques, simulated annealing, and other enhanced sampling methods including replica-exchange simulations. IDA also houses a collection of molecule-specific force fields (FFs) and experimental datasets. Uploaded MD outputs, FFs, and experimental data are integrated in a standardized format, allowing efficient data mining and extraction of valuable insights from the extensive data generated by diverse MD simulations. With the data and metadata holdings of IDA, and the prospective assignment of persistent identifiers, our work aims to make key steps towards making MD data FAIR (findable, accessible, interoperable, reusable).

bioinformatics↗

bioRxiv2024

Can We Ever Develop an Ideal RNA Force Field? Lessons Learned from Simulations of UUCG RNA Tetraloop and Other Systems.

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, DESAMBER, and OL3R2.7, successfully preserved the native structure over a 10 x 20 {micro}s timescale. 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 ({Delta}G{degrees}fold) varied significantly among these three ffs, with values of 0.0 {+/-} 0.6 kcal/mol, 2.4 {+/-} 0.8 kcal/mol, and 7.4 {+/-} 0.2 kcal/mol for OL3CP-gHBfix21, DESAMBER, and OL3R2.7, respectively. The {Delta}G{degrees}fold value from OL3CP-gHBfix21 was closest to experimental data, while the higher {Delta}G{degrees}fold values from DESAMBER and OL3R2.7 were unexpected, suggesting an over- or underestimation of key interactions within the folded and mis(un)folded ensembles. These discrepancies led us to further test DESAMBER 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 motifs 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.

biophysics↗

bioRxiv2024

How Binding Site Flexibility Promotes RNA Scanning in TbRGG2 RRM: A Molecular Dynamics Simulation Study

RNA Recognition Motifs (RRMs) are a key class of proteins that primarily bind single-stranded RNAs. In this study, we use unbiased molecular dynamics simulations to obtain insights into the intricate binding dynamics between uridine-rich RNAs and TbRGG2 RRM. Complementing structural experiments that unveil a primary binding mode with a single uridine bound, our simulations uncover two supplementary binding modes where adjacent nucleotides encroach upon the binding pocket. This leads to a unique molecular mechanism through which TbRGG2 RRM is capable of rapidly transitioning the U-rich sequence. In contrast, presence of non-native cytidines induces stalling and destabilization of the complex. By leveraging extensive equilibrium dynamics and large variety of binding states, TbRGG2 RRM effectively expedites diffusion along the RNA substrate while ensuring robust selectivity for U-rich sequences despite featuring a solitary binding pocket. Using recently developed Stafix potential, we substantiate our description of the complex dynamics by simulating fully spontaneous association process of U-rich sequences to the TbRGG2 RRM. Our study highlights critical role of dynamics and auxiliary binding states in interface dynamics employed by RNA-binding proteins, which is not readily apparent in traditional structural studies, but could represent a general type of binding strategy employed by many RNA-binding proteins.

biophysics↗

bioRxiv2024

Transcriptome-scale analysis uncovers conserved residues in the hydrophobic core of the bacterial RNA chaperone Hfq required for small regulatory RNA stability

The RNA-chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal molecular architecture of the Hfq hexamer contains three well characterised surfaces which allow it to bind sRNAs to stabilise them and engage target transcripts. Hfq-interacting sRNAs are categorised into two classes based on the surfaces they use to bind Hfq. By characterising a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen starvation, we corroborated the important role of the three RNA binding surfaces for Hfq function. Surprisingly, we uncovered two conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfqs RNA binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilisation of both sRNA classes. However, the alanine substitutions at these residues had no measurable impact on protein stability, structure or equilibrium binding to target sRNAs in vitro. We propose that V22 and G34 are key to the cooperative function among the RNA-binding surfaces of Hfq, a mechanism especially critical under cellular conditions when there is an increased demand for Hfq.

microbiology↗

bioRxiv2024

Refinement of the Sugar Puckering Torsion Potential in the AMBER DNA Force Field

The transition from B-DNA to A-DNA occurs in many protein-DNA interactions or in DNA/RNA hybrid duplexes, and thus plays a role in many important biomolecular processes that convey the biological function of DNA. However, the stability of A-DNA is severely underestimated in current AMBER force fields such as OL15, OL21 or bsc1, potentially leading to unstable or deformed protein-DNA complexes. In this study, we refine the deoxyribose dihedral potential to increase the stability of the north (N) puckering present in A-DNA. The new parameters, termed OL24, model A/B equilibrium in B-DNA duplexes in water in good agreement with NMR experiment. They also improve the description of DNA/RNA hybrids and the transition of the DNA duplex to the A-form in concentrated ethanol solutions. These refinements significantly improve the modeling of protein-DNA complexes, increasing their structural stability and A-form population, while maintaining accurate representation of canonical B-DNA duplexes. Overall, the new parameters should allow more reliable modeling of the thermodynamic equilibrium between A- and B-DNA forms and the interactions of DNA with proteins.

biophysics↗

bioRxiv2024

Comprehensive Assessment of Force-Field Performance in Molecular Dynamics Simulations of DNA/RNA Hybrid Duplexes

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 hybrids 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 the hybrids 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 hybrids. The AMBER force fields are unable to populate the C3'-endo (north) pucker of the DNA strand and underestimate inclination. 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, as well as highlight potential pitfalls and best practices for obtaining optimal performance.

biophysics↗

bioRxiv2024

The Drosophila RNA binding protein Hrp48 binds a specificRNA sequence of the msl-2 mRNA 3' UTR to regulatetranslation

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3 untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3 UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.

biophysics↗

bioRxiv2024

Mechanical stability and unfolding pathways of parallel tetrameric G-quadruplexes probed by pulling simulations

Guanine-quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, many aspects of which remain elusive, despite being important for understanding of structure formation and biological functions of GQs. Pulling experiments are a common tool for acquiring insights into the folding landscape of GQs. Herein, we applied a computational pulling strategy - Steered Molecular Dynamics (SMD) simulations - combined with standard Molecular Dynamics (MD) simulations to explore the unfolding landscapes of tetrameric parallel GQs. We identified anisotropic properties of elastic conformational changes, unfolding transitions, and GQ mechanical stabilities. The vertical component of pulling force (perpendicular to the average G-quartet plane) plays a significant role in disrupting GQ structures and weakening their mechanical stabilities. We demonstrated that the magnitude of vertical force component depends on the pulling anchor positions and the number of G-quartets. Typical unfolding transitions for tetrameric parallel GQs involve base unzipping, opening of the G-stem, strand slippage and rotation to cross-like structures. The unzipping was detected as the first and dominant unfolding event and it usually started at the 3-end. Furthermore, results from both SMD and standard MD simulations indicate that partial spiral conformations serve as a transient ensemble during the (un)folding of GQs.

biophysics↗

bioRxiv2023

Parallel G-quadruplex folds via multiple paths involving G-tract stacking and structuring from coil ensemble

G-quadruplexes (G4s) are non-canonical nucleic acid structures that fold through complex processes. Characterization of G4 folding landscape contributes to comprehending G4 roles in gene regulation but is challenging for experiments and computations. Here we investigate the folding of a three-quartet parallel DNA G4 with (GGGA)3GGG sequence using all-atom explicit-solvent enhanced-sampling molecular dynamics (MD) simulations. We suggest an early formation of guanine stacks in the G-tracts, which behave as semi-rigid blocks in the folding process. The parallel G4 folding is initiated by the formation of a collapsed compact coil-like ensemble. Structuring of the G4 from the coil then proceeds via various cross-like, hairpin, slip-stranded, and two-quartet ensembles and can bypass the G-triplex structure. While parallel G-hairpins are extremely unstable when isolated, they are more stable inside the coil structure. Folding of parallel G4 does not appear to involve any salient intermediates and, instead, it is an extremely multiple-pathway process. On the methodology side, we show that the AMBER DNA force field predicts the folded G4 to be less stable than the unfolded ensemble, uncovering substantial force-field issues. Overall, we provide unique atomistic insights into the folding landscape of parallel-stranded G4 but also reveal limitations of the state-of-the-art MD techniques.

molecular biology↗

bioRxiv2023

Simple Adjustment of Intra-nucleotide Base-phosphate Interaction in OL3 AMBER Force Field Improves RNA Simulations

Molecular dynamics (MD) simulations represent an established tool to study RNA molecules. Outcome of MD studies depends, however, on the quality of the used force field (ff). Here we suggest a correction for the widely used AMBER OL3 ff by adding a simple adjustment of nonbonded parameters. The reparameterization of Lennard-Jones potential for the -H8...O5- and -H6...O5- atom pairs addresses an intra-nucleotide steric clash occurring in the type 0 base-phosphate interaction (0BPh). The non-bonded fix (NBfix) modification of 0BPh interactions (the NBfix0BPh modification) was tuned via reweighting approach and, subsequently, tested using extensive set of standard and enhanced sampling simulations of both unstructured and folded RNA motifs. The modification corrects minor but visible intra-nucleotide 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.

biophysics↗