Biology subjects

Stadlbauer, P.

Publications and source records attributed to Stadlbauer, P..

11 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↗

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

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

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↗

bioRxiv2023

Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations

Guanine quadruplexes (GQs) are non-canonical nucleic acid structures involved in many biological processes. GQs formed in single-stranded regions often need to be unwound by cellular machinery, so their mechanochemical properties are important. Here, we performed steered molecular dynamics simulations of human telomeric GQs to study their unfolding. We examined four pulling regimes, including very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism. The more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened. If the GQ had either all-anti or all-syn pattern in a strand, strand slippage mechanism was more likely to occur. Importantly, slower pulling velocity led to richer unfolding pathways including partial refolding attempts. We show that GQ may eventually unfold after force drop under forces smaller than those the GQ withstood before the drop. This suggests that proteins in vivo might resolve GQs even if their stall forces are smaller than GQ rupture force. Finally, we found out that different unfolding intermediates may have very similar chain end-to-end distance, which reveals some limitations of structural interpretations of single-molecule spectroscopic data.

biophysics↗

bioRxiv2022

Spontaneous binding of single-stranded RNAs to RRM proteins visualised by unbiased atomistic simulations with rescaled RNA force field

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 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 (MD) 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 millisecond-scale aggregate ensemble of unbiased simulations we were able to observe a few dozens of binding events. The 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. The SRFS1 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 vdW interactions of the RNA which also improves description of the RNA-RRM bound state. Our study opens a new avenue for large-scale atomistic investigations of binding landscapes of protein-RNA complexes and future perspectives of such research are discussed.

molecular biology↗

bioRxiv2020

Insights into G-Quadruplex-Hemin Dynamics Using Atomistic Simulations: Implications for Reactivity and Folding

Guanine quadruplex nucleic acids (G4s) are involved in key biological processes such as replication or transcription. Beyond their biological relevance, G4s find applications as biotechnological tools since they readily bind hemin and enhance its peroxidase activity, creating a G4-DNAzyme. The biocatalytic properties of G4-DNAzymes have been thoroughly studied and used for biosensing purposes. Despite hundreds of applications and massive experimental efforts, the atomistic details of the reaction mechanism remain unclear. To help select between the different hypotheses currently under investigation, we use extended explicit-solvent molecular dynamics (MD) simulations to scrutinize the G4/hemin interaction. We find that besides the dominant conformation in which hemin is stacked atop the external G-quartets, hemin can also transiently bind to the loops and be brought to the external G-quartets through diverse delivery mechanisms. The simulations do not support the catalytic mechanism relying on a wobbling guanine. Similarly, catalytic role of the iron-bound water molecule is not in line with our results, however, given the simulation limitations, this observation should be considered with some caution. The simulations rather suggest tentative mechanisms in which the external G-quartet itself could be responsible for the unique H2O2-promoted biocatalytic properties of the G4/hemin complexes. Once stacked atop a terminal G-quartet, hemin rotates about its vertical axis while readily sampling shifted geometries where the iron transiently contacts oxygen atoms of the adjacent G-quartet. This dynamics is not apparent from the ensemble-averaged structure. We also visualize transient interactions between the stacked hemin and the G4 loops. Finally, we investigated interactions between hemin and on-pathway folding intermediates of the parallel-stranded G4 fold. The simulations suggest that hemin drives the folding of parallel-stranded G4s from slip-stranded intermediates, acting as a G4 chaperone. Limitations of the MD technique are briefly discussed. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/375691v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@384380org.highwire.dtl.DTLVardef@1d7c730org.highwire.dtl.DTLVardef@1562248org.highwire.dtl.DTLVardef@1e4cb9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Primitive RNA-catalysis with guanine-rich oligonucleotide sequences - the case of a (GGC)3 nonamer

A cornerstone of molecular evolution leading to the emergence of life on our planet is associated with appearance of the first catalytic RNA molecules. A question remains regarding the nature of the simplest catalytic centers that could mediate the chemistry needed for RNA-catalysis. In the current paper we provide a new example supporting our previously suggested model proposing that transiently formed open loop geometries could serve as temporary catalytic sites in the most ancient short oligonucleotides. In particular, using two independent detection techniques, PAGE and MALDI-ToF analysis, we show that prolonged thermal treatment of a 5-phosphorylated (GGC)3 sequence at weakly acidic or neutral pH in the presence of tris(hydroxymethyl)aminomethane, produces a species characterized by a (GGC)3G stoichiometry, which is compatible with the cleavage-terminal recombination chemistry suggested in our previous studies. Our new findings are complemented by microsecond-scale molecular dynamics simulations, showing that (GGC)3 dimers readily sample transient potentially catalytic geometries compatible with the experimentally observed terminal recombination chemistry.

biochemistry↗

bioRxiv2019

Stability of Two-quartet G-quadruplexes and Their Dimers in Atomistic Simulations

G-quadruplexes (GQs) are four-stranded non-canonical DNA and RNA architectures that can be formed by guanine-rich sequences. The stability of GQs increases with the number of G-quartets and three G-quartets generally form stable GQs. However, the stability of two-quartet GQs is an open issue. To understand the intrinsic stability of two-quartet GQ stems, we have carried out a series of unbiased molecular dynamics (MD) simulations ([~]505 {micro}s in total) of two- and four-quartet DNA and RNA GQs, with attention paid mainly to parallel-stranded arrangements. We used AMBER DNA parmOL15 and RNA parmOL3 force fields and tested different ion and water models. DNA two-quartet parallel-stranded GQs unfolded in all the simulations while the equivalent RNA GQ was stable in most of the simulations. GQs composed of two stacked units of two-quartet GQs were stable for both DNA and RNA. The simulations suggest that a minimum of three quartets are needed to form an intrinsically stable all-anti parallel-stranded DNA GQ. Parallel two-quartet DNA GQ may exist if substantially stabilized by another molecule or structural element, including multimerisation. On the other hand, we predict that isolated RNA two-quartet parallel GQs may form, albeit being weakly stable. We also show that ionic parameters and water models should be chosen with caution because some parameter combinations can cause spurious instability of GQ stems. Some in-so-far unnoticed limitations of force-field description of multiple ions inside the GQs are discussed, which compromise capability of simulations to fully capture the effect of increase of the number of quartets on the GQ stability.

biophysics↗