Guanine quadruplexes (GQs) play crucial roles in various biological processes, and understanding their folding pathways provides insight into their stability, dynamics, and functions. This knowledge aids in designing therapeutic strategies, as GQs are potential targets for anticancer drugs and other therapeutics. Although experimental and theoretical techniques have provided valuable insights into different stages of the GQ folding, the structural complexity of GQs poses significant challenges, and our understanding remains incomplete. This study introduces a novel computational protocol for folding an entire GQ from single-strand conformation to its native state. By combining two complementary enhanced sampling techniques, we were able to model folding pathways, encompassing a diverse range of intermediates. Although our investigation of the GQ free energy surface (FES) is focused solely on the folding of the all-anti parallel GQ topology, this protocol has the potential to be adapted for the folding of systems with more complex folding landscapes.

• Keywords
• DNA quadruplex, computational folding, enhanced sampling, kinetic partitioning mechanism, metadynamics, molecular dynamics, nudged elastic band, pathCV, transition path sampling,
• MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Nucleic Acid Conformation MeSH
• Molecular Dynamics Simulation MeSH
• Thermodynamics MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH

G-quadruplexes (G4 s), as non-canonical DNA structures, attract a great deal of research interest in the molecular biology as well as in the material science fields. The use of small molecules as ligands for G-quadruplexes has emerged as a tool to regulate gene expression and telomeres maintenance. Meso-tetrakis-(N-methyl-4-pyridyl) porphyrin (TMPyP4) was shown as one of the first ligands for G-quadruplexes and it is still widely used. We report an investigation comprising molecular docking and dynamics, synthesis and multiple spectroscopic and spectrometric determinations on simple cationic porphyrins and their interaction with different DNA sequences. This study enabled the synthesis of tetracationic porphyrin derivatives that exhibited binding and stabilizing capacity against G-quadruplex structures; the detailed characterization has shown that the presence of amide groups at the periphery improves selectivity for parallel G4 s binding over other structures. Taking into account the ease of synthesis, 5,10,15,20-tetrakis-(1-acetamido-4-pyridyl) porphyrin bromide could be considered a better alternative to TMPyP4 in studies involving G4 binding.

• Keywords
• DNA, G-quadruplexes, Molecular dynamics, Molecular recognition, NMR,
• MeSH
• Circular Dichroism MeSH
• DNA * chemistry   MeSH
• G-Quadruplexes * MeSH
• Ligands MeSH
• Porphyrins * chemistry   MeSH
• Molecular Dynamics Simulation MeSH
• Molecular Docking Simulation * MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA * MeSH
• Ligands MeSH
• Porphyrins * MeSH
• tetra(4-N-methylpyridyl)porphine MeSH Browser

Guanine quadruplex (GQ) is a noncanonical nucleic acid structure formed by guanine-rich DNA and RNA sequences. Folding of GQs is a complex process, where several aspects remain elusive, despite being important for understanding 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─in combination 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. Using a special set of structural parameters, we found that 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 the 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.

• MeSH
• Biomechanical Phenomena MeSH
• DNA chemistry   MeSH
• G-Quadruplexes * MeSH
• Mechanical Phenomena MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH

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 a very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism, i.e.,: (i) the more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened, (ii) the more parallel the direction of force was, GQ opening and cross-like GQs were more likely to occur, (iii) strand slippage mechanism was possible for GQs with an all-anti pattern in a strand, and (iv) slower pulling velocity led to richer structural dynamics with sampling of more intermediates and partial refolding events. We also identified that a GQ may eventually unfold after a force drop under forces smaller than those that the GQ withstood before the drop. Finally, we found out that different unfolding intermediates could have very similar chain end-to-end distances, which reveals some limitations of structural interpretations of single-molecule spectroscopic data.

• MeSH
• G-Quadruplexes * MeSH
• Guanine * chemistry   MeSH
• Humans MeSH
• Mechanical Phenomena MeSH
• Molecular Dynamics Simulation MeSH
• Telomere MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Guanine * 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

Recently, we reported an inhibitory effect of guanine substitutions on the conformational switch from antiparallel to parallel quadruplexes (G4) induced by dehydrating agents. As a possible cause, we proposed a difference in the sensitivity of parallel and antiparallel quadruplexes to the guanine substitutions in the resulting thermodynamic stability. Reports on the influence of guanine substitutions on the biophysical properties of intramolecular parallel quadruplexes are rare. Moreover, such reports are often complicated by the multimerisation tendencies of parallel quadruplexes. To address this incomplete knowledge, we employed circular dichroism spectroscopy (CD), both as stopped-flow-assisted fast kinetics measurements and end-point measurements, accompanied by thermodynamic analyses, based on UV absorption melting profiles, and electrophoretic methods. We showed that parallel quadruplexes are significantly more sensitive towards guanine substitutions than antiparallel ones. Furthermore, guanine-substituted variants, which in principle might correspond to native genomic sequences, distinctly differ in their biophysical properties, indicating that the four guanines in each tetrad of parallel quadruplexes are not equal. In addition, we were able to distinguish by CD an intramolecular G4 from intermolecular ones resulting from multimerisation mediated by terminal tetrad association, but not from intermolecular G4s formed due to inter-strand Hoogsteen hydrogen bond formation. In conclusion, our study indicates significant variability in parallel quadruplex structures, otherwise disregarded without detailed experimental analysis.

• Keywords
• DNA secondary structure, circular dichroism, multimerisation, parallel guanine quadruplex, stopped-flow,
• MeSH
• Circular Dichroism MeSH
• DNA chemistry   genetics   MeSH
• G-Quadruplexes MeSH
• Guanine chemistry   MeSH
• Nucleic Acid Conformation MeSH
• Models, Molecular MeSH
• Amino Acid Substitution * MeSH
• Thermodynamics MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Guanine MeSH

Guanine quadruplexes (G4s) are non-canonical nucleic acids structures common in important genomic regions. Parallel-stranded G4 folds are the most abundant, but their folding mechanism is not fully understood. Recent research highlighted that G4 DNA molecules fold via kinetic partitioning mechanism dominated by competition amongst diverse long-living G4 folds. The role of other intermediate species such as parallel G-triplexes and G-hairpins in the folding process has been a matter of debate. Here, we use standard and enhanced-sampling molecular dynamics simulations (total length of ∼0.9 ms) to study these potential folding intermediates. We suggest that parallel G-triplex per se is rather an unstable species that is in local equilibrium with a broad ensemble of triplex-like structures. The equilibrium is shifted to well-structured G-triplex by stacked aromatic ligand and to a lesser extent by flanking duplexes or nucleotides. Next, we study propeller loop formation in GGGAGGGAGGG, GGGAGGG and GGGTTAGGG sequences. We identify multiple folding pathways from different unfolded and misfolded structures leading towards an ensemble of intermediates called cross-like structures (cross-hairpins), thus providing atomistic level of description of the single-molecule folding events. In summary, the parallel G-triplex is a possible, but not mandatory short-living (transitory) intermediate in the folding of parallel-stranded G4.

• MeSH
• DNA chemistry   genetics   metabolism   MeSH
• G-Quadruplexes * MeSH
• Guanine chemistry   metabolism   MeSH
• DNA, Single-Stranded chemistry   genetics   metabolism   MeSH
• Kinetics MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Base Sequence MeSH
• Molecular Dynamics Simulation * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• Guanine MeSH
• DNA, Single-Stranded MeSH

Molecular dynamics (MD) simulations became a leading tool for investigation of structural dynamics of nucleic acids. Despite recent efforts to improve the empirical potentials (force fields, ffs), RNA ffs have persisting deficiencies, which hamper their utilization in quantitatively accurate simulations. Previous studies have shown that at least two salient problems contribute to difficulties in the description of free-energy landscapes of small RNA motifs: (i) excessive stabilization of the unfolded single-stranded RNA ensemble by intramolecular base-phosphate and sugar-phosphate interactions and (ii) destabilization of the native folded state by underestimation of stability of base pairing. Here, we introduce a general ff term (gHBfix) that can selectively fine-tune nonbonding interaction terms in RNA ffs, in particular, the H bonds. The gHBfix potential affects the pairwise interactions between all possible pairs of the specific atom types, while all other interactions remain intact; i.e., it is not a structure-based model. In order to probe the ability of the gHBfix potential to refine the ff nonbonded terms, we performed an extensive set of folding simulations of RNA tetranucleotides and tetraloops. On the basis of these data, we propose particular gHBfix parameters to modify the AMBER RNA ff. The suggested parametrization significantly improves the agreement between experimental data and the simulation conformational ensembles, although our current ff version still remains far from being flawless. While attempts to tune the RNA ffs by conventional reparametrizations of dihedral potentials or nonbonded terms can lead to major undesired side effects, as we demonstrate for some recently published ffs, gHBfix has a clear promising potential to improve the ff performance while avoiding introduction of major new imbalances.

• MeSH
• RNA chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA MeSH

We have carried out an extended set of standard and enhanced-sampling MD simulations (for a cumulative simulation time of 620 μs) with the aim to study folding landscapes of the rGGGUUAGGG and rGGGAGGG parallel G-hairpins (PH) with propeller loop. We identify folding and unfolding pathways of the PH, which is bridged with the unfolded state via an ensemble of cross-like structures (CS) possessing mutually tilted or perpendicular G-strands interacting via guanine-guanine H-bonding. The oligonucleotides reach the PH conformation from the unfolded state via a conformational diffusion through the folding landscape, i.e. as a series of rearrangements of the H-bond interactions starting from compacted anti-parallel hairpin-like structures. Although isolated PHs do not appear to be thermodynamically stable we suggest that CS and PH-types of structures are sufficiently populated during RNA guanine quadruplex (GQ) folding within the context of complete GQ-forming sequences. These structures may participate in compact coil-like ensembles that involve all four G-strands and already some bound ions. Such ensembles can then rearrange into the fully folded parallel GQs via conformational diffusion. We propose that the basic atomistic folding mechanism of propeller loops suggested in this work may be common for their formation in RNA and DNA GQs.

• MeSH
• G-Quadruplexes * MeSH
• Guanine chemistry   metabolism   MeSH
• Kinetics MeSH
• RNA chemistry   metabolism   MeSH
• RNA Folding * MeSH
• Base Sequence MeSH
• Molecular Dynamics Simulation MeSH
• Thermodynamics MeSH
• Hydrogen Bonding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Guanine MeSH
• RNA MeSH

With both catalytic and genetic functions, ribonucleic acid (RNA) is perhaps the most pluripotent chemical species in molecular biology, and its functions are intimately linked to its structure and dynamics. Computer simulations, and in particular atomistic molecular dynamics (MD), allow structural dynamics of biomolecular systems to be investigated with unprecedented temporal and spatial resolution. We here provide a comprehensive overview of the fast-developing field of MD simulations of RNA molecules. We begin with an in-depth, evaluatory coverage of the most fundamental methodological challenges that set the basis for the future development of the field, in particular, the current developments and inherent physical limitations of the atomistic force fields and the recent advances in a broad spectrum of enhanced sampling methods. We also survey the closely related field of coarse-grained modeling of RNA systems. After dealing with the methodological aspects, we provide an exhaustive overview of the available RNA simulation literature, ranging from studies of the smallest RNA oligonucleotides to investigations of the entire ribosome. Our review encompasses tetranucleotides, tetraloops, a number of small RNA motifs, A-helix RNA, kissing-loop complexes, the TAR RNA element, the decoding center and other important regions of the ribosome, as well as assorted others systems. Extended sections are devoted to RNA-ion interactions, ribozymes, riboswitches, and protein/RNA complexes. Our overview is written for as broad of an audience as possible, aiming to provide a much-needed interdisciplinary bridge between computation and experiment, together with a perspective on the future of the field.

• MeSH
• DNA chemistry   MeSH
• Catalysis MeSH
• Nucleic Acid Conformation * MeSH
• Computer Simulation MeSH
• RNA chemistry   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA MeSH
• RNA MeSH