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.

• Klíčová slova
• DNA quadruplex, computational folding, enhanced sampling, kinetic partitioning mechanism, metadynamics, molecular dynamics, nudged elastic band, pathCV, transition path sampling,
• MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny MeSH
• simulace molekulární dynamiky MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 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-kvadruplexy * MeSH
• guanin * chemie   MeSH
• lidé MeSH
• mechanické jevy MeSH
• simulace molekulární dynamiky MeSH
• telomery MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• guanin * 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
• konformace nukleové kyseliny MeSH
• kruhová DNA chemie   MeSH
• molekulární modely MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• nukleotidové motivy MeSH
• párování bází MeSH
• Saccharomyces cerevisiae genetika   MeSH
• stereoizomerie MeSH
• telomery chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kruhová DNA 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 chemie   genetika   metabolismus   MeSH
• G-kvadruplexy * MeSH
• guanin chemie   metabolismus   MeSH
• jednovláknová DNA chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• konformace nukleové kyseliny * MeSH
• lidé MeSH
• sekvence nukleotidů MeSH
• simulace molekulární dynamiky * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• guanin MeSH
• jednovláknová DNA 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-kvadruplexy * MeSH
• guanin chemie   metabolismus   MeSH
• kinetika MeSH
• RNA chemie   metabolismus   MeSH
• sbalování RNA * MeSH
• sekvence nukleotidů MeSH
• simulace molekulární dynamiky MeSH
• termodynamika MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• guanin MeSH
• RNA MeSH

We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory.

• MeSH
• DNA chemie   genetika   metabolismus   MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• sekvence nukleotidů MeSH
• shluková analýza MeSH
• simulace molekulární dynamiky * MeSH
• telomery genetika   MeSH
• voda chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• voda MeSH