Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations

Explicit solvent molecular dynamics simulations have been used to complement preceding experimental and computational studies of folding of guanine quadruplexes (G-DNA). We initiate early stages of unfolding of several G-DNAs by simulating them under no-salt conditions and then try to fold them back using standard excess salt simulations. There is a significant difference between G-DNAs with all-anti parallel stranded stems and those with stems containing mixtures of syn and anti guanosines. The most natural rearrangement for all-anti stems is a vertical mutual slippage of the strands. This leads to stems with reduced numbers of tetrads during unfolding and a reduction of strand slippage during refolding. The presence of syn nucleotides prevents mutual strand slippage; therefore, the antiparallel and hybrid quadruplexes initiate unfolding via separation of the individual strands. The simulations confirm the capability of G-DNA molecules to adopt numerous stable locally and globally misfolded structures. The key point for a proper individual folding attempt appears to be correct prior distribution of syn and anti nucleotides in all four G-strands. The results suggest that at the level of individual molecules, G-DNA folding is an extremely multi-pathway process that is slowed by numerous misfolding arrangements stabilized on highly variable timescales.     

Hairpins participating in folding of human telomeric sequence quadruplexes studied by standard and T-REMD simulations

DNA G-hairpins are potential key structures participating in folding of human telomeric guanine quadruplexes (GQ). We examined their properties by standard MD simulations starting from the folded state and long T-REMD starting from the unfolded state, accumulating ∼130 μs of atomistic simulations. Antiparallel G-hairpins should spontaneously form in all stages of the folding to support lateral and diagonal loops, with sub-μs scale rearrangements between them. We found no clear predisposition for direct folding into specific GQ topologies with specific syn/anti patterns. Our key prediction stemming from the T-REMD is that an ideal unfolded ensemble of the full GQ sequence populates all 4096 syn/anti combinations of its four G-stretches. The simulations can propose idealized folding pathways but we explain that such few-state pathways may be misleading. In the context of the available experimental data, the simulations strongly suggest that the GQ folding could be best understood by the kinetic partitioning mechanism with a set of deep competing minima on the folding landscape, with only a small fraction of molecules directly folding to the native fold. The landscape should further include non-specific collapse processes where the molecules move via diffusion and consecutive random rare transitions, which could, e.g. structure the propeller loops.     

Cation binding to 15-TBA quadruplex DNA is a multiple-pathway cation-dependent process

A combination of explicit solvent molecular dynamics simulation (30 simulations reaching 4 μs in total), hybrid quantum mechanics/molecular mechanics approach and isothermal titration calorimetry was used to investigate the atomistic picture of ion binding to 15-mer thrombin-binding quadruplex DNA (G-DNA) aptamer. Binding of ions to G-DNA is complex multiple pathway process, which is strongly affected by the type of the cation. The individual ion-binding events are substantially modulated by the connecting loops of the aptamer, which play several roles. They stabilize the molecule during time periods when the bound ions are not present, they modulate the route of the ion into the stem and they also stabilize the internal ions by closing the gates through which the ions enter the quadruplex. Using our extensive simulations, we for the first time observed full spontaneous exchange of internal cation between quadruplex molecule and bulk solvent at atomistic resolution. The simulation suggests that expulsion of the internally bound ion is correlated with initial binding of the incoming ion. The incoming ion then readily replaces the bound ion while minimizing any destabilization of the solute molecule during the exchange.     

Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K⁺. The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel–antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5′- and 3′-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K⁺. Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K⁺. The unique human telomeric G-quadruplex structure formed in K⁺ suggests that it can be specifically targeted for anticancer drug design.     

Fast-Folding Pathways of the Thrombin-Binding Aptamer G-Quadruplex Revealed by a Markov State Model

G-quadruplex structures participate in many important cellular processes. For a better understanding of their functions, knowledge of the mechanism by which they fold into the functional native structures is necessary. In this work, we studied the folding process of the thrombin-binding aptamer G-quadruplex. Enabled by a computational paradigm that couples an advanced sampling method and a Markov state model, four folding intermediates were identified, including an antiparallel G-hairpin, two G-triplex structures, and a double-hairpin conformation. Likewise, a misfolded structure with a nonnative distribution of syn/anti guanines was also observed. Based on these states, a transition path analysis revealed three fast-folding pathways, along which the thrombin-binding aptamer would fold to the native state directly, with no evidence of potential nonnative competing conformations. The results also showed that the TGT-loop plays an important role in the folding process. The findings of this research may provide general insight about the folding of other G-quadruplex structures.     

Strand directionality affects cation binding and movement within tetramolecular G-quadruplexes

Nuclear magnetic resonance study of G-quadruplex structures formed by d(TG₃T) and its modified analogs containing a 5′-5′ or 3′-3′ inversion of polarity sites, namely d(3′TG5′-5′G₂T3′), d(3′T5′-5′G₃T3′) and d(5′TG3′-3′G₂T5’) demonstrates formation of G-quadruplex structures with tetrameric topology and distinct cation-binding preferences. All oligonucleotides are able to form quadruplex structures with two binding sites, although the modified oligonucleotides also form, in variable amounts, quadruplex structures with only one bound cation. The inter-quartet cavities at the inversion of polarity sites bind ammonium ions less tightly than a naturally occurring 5′-3′ backbone. Exchange of ¹⁵ ions between G-quadruplex and bulk solution is faster at the 3′-end in comparison to the 5′-end. In addition to strand directionality, cation movement is influenced by formation of an all-syn G-quartet. Formation of such quartet has been observed also for the parent d(TG₃T) that besides the canonical quadruplex with only all-anti G-quartets, forms a tetramolecular parallel quadruplex containing one all-syn G-quartet, never observed before in unmodified quadruplex structures.     

Formation pathways of a guanine-quadruplex DNA revealed by molecular dynamics and thermodynamic analysis of the substates

The formation of a cation-stabilized guanine quadruplex (G-DNA) stem is an exceptionally slow process involving complex kinetics that has not yet been characterized at atomic resolution. Here, we investigate the formation of a parallel stranded G-DNA stem consisting of four strands of d(GGGG) using molecular dynamics simulations with explicit inclusion of counterions and solvent. Due to the limitations imposed by the nanosecond timescale of the simulations, rather than watching for the spontaneous formation of G-DNA, our approach probes the stability of possible supramolecular intermediates (including two-, three-, and four-stranded assemblies with out-of-register base pairing between guanines) on the formation pathway. The simulations suggest that "cross-like" two-stranded assemblies may serve as nucleation centers in the initial formation of parallel stranded G-DNA quadruplexes, proceeding through a series of rearrangements involving trapping of cations, association of additional strands, and progressive slippage of strands toward the full stem. To supplement the analysis, approximate free energies of the models are obtained with explicit consideration of the integral cations. The approach applied here serves as a prototype for qualitatively investigating other G-DNA molecules using molecular dynamics simulation and free-energy analysis.     

A novel chair-type G-quadruplex formed by a Bombyx mori telomeric sequence

Recently, the human telomeric d[TAGGG(TTAGGG)₃] sequence has been shown to form in K⁺ solution an intramolecular (3+1) G-quadruplex structure, whose G-tetrad core contains three strands oriented in one direction and the fourth in the opposite direction. Here we present a study on the structure of the Bombyx mori telomeric d[TAGG(TTAGG)₃] sequence, which differs from the human counterpart only by one G deletion in each repeat. We found that this sequence adopted multiple G-quadruplex structures in K⁺ solution. We have favored a major G-quadruplex form by a judicious U-for-T substitution in the sequence and determined the folding topology of this form. We showed by NMR that this was a new chair-type intramolecular G-quadruplex which involved a two-layer antiparallel G-tetrad core and three edgewise loops. Our result highlights the effect of G-tract length on the folding topology of G-quadruplexes, but also poses the question of whether a similar chair-type G-quadruplex fold exists in the human telomeric sequences.     

Evidence of different G-quadruplex DNA binding with biogenic polyamines probed by electrospray ionization-quadrupole time of flight mass spectrometry,circular dichroism and atomic force microscopy

The binding properties of five G-quadruplex oligonucleotides (humtel24, k-ras32, c-myc22, c-kit1 and c-kit2) with polyamines have been investigated by electrospray ionization-quadrupole time of flight mass spectrometry, circular dichroism, melting temperature, atomic force microscopy (AFM) and molecular simulation. The MS results demonstrated that the polyamines and G-quadruplex DNA can form complexes with high affinity, and one molecule of G-quadruplex DNA can combine several molecules (1–5) of polyamines. The binding affinities of the polyamines to DNA were in the order of spermine > spermidine > putrescine. After binding with polyamines, the conformations of the G-quadruplex DNA were significantly changed, and spermine can induce the configurations of k-ras32 and c-kit1 to deviate from their G-quadruplex structures at high concentrations. In the presence of K⁺, the conformations of G-quadruplex DNA were stabilized, while polyamines can also induced alterations of their configurations. Melting temperature experiments suggested that the T_m of the DNA–polyamine complexes obviously increased both in the absence and presence of K⁺. The AFM results indicated that polyamines can induce aggregation of G-quadruplex DNA. Above results illustrated that the polyamines bound with the phosphate backbone and the base-pairs of G-quadruplex structures. Combining with the molecular simulation, the binding mode of the G-quadruplex DNA and polyamines were discussed. The results obtained would be beneficial for understanding the biological and physiological functions of polyamines and provide useful information for development of antitumor drugs.     

Common Structural Transitions in Explicit-Solvent Simulations of Villin Headpiece Folding

Molecular dynamics simulations of protein folding can provide very high-resolution data on the folding process; however, due to computational challenges most studies of protein folding have been limited to small peptides, or made use of approximations such as Gō potentials or implicit solvent models. We have performed a set of molecular dynamics simulations totaling >50 μs on the villin headpiece subdomain, one of the most stable and fastest-folding naturally occurring proteins, in explicit solvent. We find that the wild-type villin headpiece reliably folds to a native conformation on timescales similar to experimentally observed folding, but that a fast folding double-norleucine mutant shows significantly more heterogeneous behavior. Along with other recent simulation studies, we note the occurrence of nonnative structures intermediates, which may yield a nativelike signal in the fluorescence measurements typically used to study villin folding. Based on the wild-type simulations, we propose alternative approaches to measure the formation of the native state.     

Isothermal folding of G-quadruplexes

Thermodynamic studies of G-quadruplex stability are an essential complement to structures obtained by NMR or X-ray crystallography. An understanding of the energetics of quadruplex folding provides a necessary foundation for the physical interpretation of quadruplex formation and reactivity. While thermal denaturation methods are most commonly used to evaluate quadruplex stability, it is also possible to study folding using isothermal titration methods. G-quadruplex folding is tightly coupled to specific cation binding. We describe here protocols for monitoring the cation-driven quadruplex folding transition using circular dichroism or absorbance, and for determination of the distribution of free and bound cation using a fluorescence indicator. Together these approaches provide insight into quadruplex folding at constant temperature, and characterize the linkage between cation binding and folding.     

All-Atom Simulations Reveal How Single-Point Mutations Promote Serpin Misfolding

Protein misfolding is implicated in many diseases, including serpinopathies. For the canonical inhibitory serpin α₁-antitrypsin, mutations can result in protein deficiencies leading to lung disease, and misfolded mutants can accumulate in hepatocytes, leading to liver disease. Using all-atom simulations based on the recently developed bias functional algorithm, we elucidate how wild-type α₁-antitrypsin folds and how the disease-associated S (Glu264Val) and Z (Glu342Lys) mutations lead to misfolding. The deleterious Z mutation disrupts folding at an early stage, whereas the relatively benign S mutant shows late-stage minor misfolding. A number of suppressor mutations ameliorate the effects of the Z mutation, and simulations on these mutants help to elucidate the relative roles of steric clashes and electrostatic interactions in Z misfolding. These results demonstrate a striking correlation between atomistic events and disease severity and shine light on the mechanisms driving chains away from their correct folding routes.     

Understanding the Role of Three-Dimensional Topology in Determining the?Folding Intermediates of Group I Introns

Many RNA molecules exert their biological function only after folding to unique three-dimensional structures. For long, noncoding RNA molecules, the complexity of finding the native topology can be a major impediment to correct folding to the biologically active structure. An RNA molecule may fold to a near-native structure but not be able to continue to the correct structure due to a topological barrier such as crossed strands or incorrectly stacked helices. Achieving the native conformation thus requires unfolding and refolding, resulting in a long-lived intermediate. We investigate the role of topology in the folding of two phylogenetically related catalytic group I introns, the Twort and Azoarcus group I ribozymes. The kinetic models describing the Mg²⁺-mediated folding of these ribozymes were previously determined by time-resolved hydroxyl (⋅OH) radical footprinting. Two intermediates formed by parallel intermediates were resolved for each RNA. These data and analytical ultracentrifugation compaction analyses are used herein to constrain coarse-grained models of these folding intermediates as we investigate the role of nonnative topology in dictating the lifetime of the intermediates. Starting from an ensemble of unfolded conformations, we folded the RNA molecules by progressively adding native constraints to subdomains of the RNA defined by the ⋅OH time-progress curves to simulate folding through the different kinetic pathways. We find that nonnative topologies (arrangement of helices) occur frequently in the folding simulations despite using only native constraints to drive the reaction, and that the initial conformation, rather than the folding pathway, is the major determinant of whether the RNA adopts nonnative topology during folding. From these analyses we conclude that biases in the initial conformation likely determine the relative flux through parallel RNA folding pathways.     

Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability

Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]₁₀ repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]₄ and [TTTTGGGG]₄ are thermally more stable compared to human [TTAGGG]₄. Consistent with this, replication of vectors with ciliate [TTGGGG]₁₀ repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]₁₀ vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.     

Conformational switch of insulin-binding aptamer into G-quadruplex induced by K+ and Na+: an experimental and theoretical approach

Guanine-rich sequences can form the G-quadruplex structure in the presence of specific metal ions. Here, circular dichroism, UV–vis absorption, fluorescence, and molecular dynamics simulation studies revealed that insulin-binding aptamer (IBA) could form an intramolecular G-quadruplex structure after binding K⁺. Circular dichroism (CD) spectra demonstrated that IBA could fold into a parallel G-quadruplex with a strong positive peak at 263?nm. Analysis of equilibrium titration data revealed that cation binding was cooperative with the Hill coefficient of 2.01 in K⁺ and 1.90 in Na⁺. Thermal denaturation assays indicated that K⁺-induced G-quadruplex is more stable than Na⁺-induced structure. Folding of IBA into G-quadruplex leading to the contact quenching occurs as a result of the formation of a nonfluorescent complex between donor and acceptor. Based on fluorescence quenching of IBA folding, a potassium-sensing aptasensor in the range of 0–1.4?mM was proposed. Since the quenching process was predominantly static, the binding constant and the number of binding sites were determined. In this research, based on the experimental data, the initial model of IBA G-quadruplex was constructed by molecular modeling method. The modeling structure of IBA is an intramolecular parallel-strand quadruplex conformation with two guanine tetrads. The extended molecular dynamics simulation for the model indicated that the G-quadruplex maintains its structure very well in aqueous solution in presence of K⁺ in the central cavity. In contrast, it was demonstrated that the G-quadruplex structure of model in the water collapses in absence of this cation.     

Extended Structures in RNA Folding Intermediates Are Due to Nonnative Interactions Rather than Electrostatic Repulsion

RNA folding occurs via a series of transitions between metastable intermediate states for Mg²⁺ concentrations below those needed to fold the native structure. In general, these folding intermediates are considerably less compact than their respective native states. Our previous work demonstrates that the major equilibrium intermediate of the 154-residue specificity domain (S-domain) of the Bacillus subtilis RNase P RNA is more extended than its native structure. We now investigate two models with falsifiable predictions regarding the origins of the extended intermediate structures in the S-domains of the B. subtilis and the Escherichia coli RNase P RNA that belong to different classes of P RNA and have distinct native structures. The first model explores the contribution of electrostatic repulsion, while the second model probes specific interactions in the core of the folding intermediate. Using small-angle X-ray scattering and Langevin dynamics simulations, we show that electrostatics plays only a minor role, whereas specific interactions largely account for the extended nature of the intermediate. Structural contacts in the core, including a nonnative base pair, help to stabilize the intermediate conformation. We conclude that RNA folding intermediates adopt extended conformations due to short-range, nonnative interactions rather than generic electrostatic repulsion of helical domains. These principles apply to other ribozymes and riboswitches that undergo functionally relevant conformational changes.     

Insight into formation propensity of pseudocircular DNA G-hairpins

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).