Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes.

Cations are known to mediate diverse interactions in nucleic acids duplexes but they are critical in the arrangement of four-stranded structures. Here, we use all-atom molecular dynamics simulations with explicit solvent to analyse the mechanical unfolding of representative intramolecular G-quadruplex structures: a parallel, a hybrid and an antiparallel DNA and a parallel RNA, in the presence of stabilising cations. We confirm the stability of these conformations in the presence of [Formula: see text] central ions and observe distortions from the tetrad topology in their absence. Force-induced unfolding dynamics is then investigated. We show that the unfolding events in the force-extension curves are concomitant to the loss of coordination between the central ions and the guanines of the G-quadruplex. We found lower ruptures forces for the parallel configuration with respect to the antiparallel one, while the behaviour of the force pattern of the parallel RNA appears similar to the parallel DNA. We anticipate that our results will be essential to interpret the fine structure rupture profiles in stretching assays at high resolution and will shed light on the mechanochemical activity of G-quadruplex-binding machinery

Unfolding mechanism and free energy landscape of single, stable, alpha helices at low pull speeds

Single alpha helices (SAHs) stable in isolated form are often found in motor proteins where they bridge functional domains. Understanding the mechanical response of SAHs is thus critical to understand their function. The quasi-static force-extension relation of a small number of SAHs is known from single-molecule experiments. Unknown, or still controversial, are the molecular scale details behind those observations. We show that the deformation mechanism of SAHs pulled from the termini at pull speeds approaching the quasi-static limit differs from that of typical helices found in proteins, which are stable only when interacting with other protein domains. Using molecular dynamics simulations with atomistic resolution at low pull speeds previously inaccessible to simulation, we show that SAHs start unfolding from the termini at all pull speeds we investigated. Unfolding proceeds residue-by-residue and hydrogen bond breaking is not the main event determining the barrier to unfolding. We use the molecular simulation data to test the cooperative Sticky Chain model. This model yields excellent fits of the force-extension curves and quantifies the distance, xE = 0.13 nm, to the transition state, the natural frequency of bond vibration, ν0 = 0.82 ns−1, and the height, V0=2.9 kcal/mol, of the free energy barrier associated with the deformation of single residues. Our results demonstrate that the Sticky Chain model could advantageously be used to analyze experimental force-extension curves of SAHs and other biopolymers

Molecular mechanics of coiled coils loaded in the shear geometry

Coiled coils are important nanomechanical building blocks in biological and biomimetic materials. A mechanistic molecular understanding of their structural response to mechanical load is essential for elucidating their role in tissues and for utilizing and tuning these building blocks in materials applications. Using a combination of single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations, we have investigated the mechanics of synthetic heterodimeric coiled coils of different length (3-4 heptads) when loaded in shear geometry. Upon shearing, we observe an initial rise in the force, which is followed by a constant force plateau and ultimately strand separation. The force required for strand separation depends on the coiled coil length and the applied loading rate, suggesting that coiled coil shearing occurs out of equilibrium. This out-of-equilibrium behaviour is determined by a complex structural response which involves helix uncoiling, uncoiling-assisted sliding of the helices relative to each other in the direction of the applied force as well as uncoiling-assisted dissociation perpendicular to the force axis. These processes follow a hierarchy of timescales with helix uncoiling being faster than sliding and sliding being faster than dissociation. In SMFS experiments, strand separation is dominated by uncoiling-assisted dissociation and occurs at forces between 25-45 pN for the shortest 3-heptad coiled coil and between 35-50 pN for the longest 4-heptad coiled coil. These values are highly similar to the forces required for shearing apart short double-stranded DNA oligonucleotides, reinforcing the potential role of coiled coils as nanomechanical building blocks in applications where protein-based structures are desired

Mesoscopic model for DNA G-quadruplex unfolding

[EN] Genomes contain rare guanine-rich sequences capable of assembling into four-stranded helical structures, termed G-quadruplexes, with potential roles in gene regulation and chromosome stability. Their mechanical unfolding has only been reported to date by all-atom simulations, which cannot dissect the major physical interactions responsible for their cohesion. Here, we propose a mesoscopic model to describe both the mechanical and thermal stability of DNA G-quadruplexes, where each nucleotide of the structure, as well as each central cation located at the inner channel, is mapped onto a single bead. In this framework we are able to simulate loading rates similar to the experimental ones, which are not reachable in simulations with atomistic resolution. In this regard, we present single-molecule force-induced unfolding experiments by a high-resolution optical tweezers on a DNA telomeric sequence capable of adopting a G-quadruplex conformation. Fitting the parameters of the model to the experiments we find a correct prediction of the rupture-force kinetics and a good agreement with previous near equilibrium measurements. Since G-quadruplex unfolding dynamics is halfway in complexity between secondary nucleic acids and tertiary protein structures, our model entails a nanoscale paradigm for non-equilibrium processes in the cell.Work supported by the Spanish Ministry of Economy and Competitiveness (MINECO), grant No. FIS2014-55867, co-financed by FEDER funds. We also thank the support of the Aragon Government and Fondo Social Europeo to FENOL group. Work in J.R.A.-G. laboratory was supported by a grant from MINECO, No. MAT2015-71806-R).Bergues-Pupo, A.; Gutiérrez, I.; Arias-Gonzalez, JR.; Falo, F.; Fiasconaro, A. (2017). Mesoscopic model for DNA G-quadruplex unfolding. Scientific Reports. 7:1-13. https://doi.org/10.1038/s41598-017-10849-2S1137Arias-Gonzalez, J. R. Single-molecule portrait of DNA and RNA double helices. Integr. Biol. 6, 904 (2014).Burge, S., Parkinson, G. N., Hazel, P., Todd, A. K. & Neidle, S. Quadruplex DNA: sequence, topology and structure. Nucleic Acids Res. 34, 5402 (2006).Lam, E. Y., Beraldi, D., Tannahill, D. & Balasubramanian, S. G-quadruplex structures are stable and detectable in human genomic DNA. Nat. Commun. 4, 1796 (2013).Siddiqui-Jain, A., Grand, C. L., Bearss, D. J. & Hurley, L. H. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc. Natl. Acad. Sci. USA 99, 11593 (2002).Endoh, T. & Sugimoto, N. Mechanical insights into ribosomal progression overcoming RNA G-quadruplex from periodical translation suppression in cells. Sci. Rep. 6, 1 (2016).Hänsel-Hertsch, R., Di Antonio, M. & Balasubramanian, S. DNA G-quadruplexes in the human genome: detection, functions and therapeutic potential. Nat. Rev. Mol. Cell Biol. 18, 279 (2017).de Messieres, M., Chang, J. C., Brawn-Cinani, B. & La Porta, A. Single-molecule study of G-quadruplex disruption using dynamic force spectroscopy. Phys. Rev. Lett. 109, 058101 (2012).Koirala, D. et al. A single-molecule platform for investigation of interactions between G-quadruplexes and small-molecule ligands. Nat. Chem. 3, 782 (2011).Long, X. et al. Mechanical unfolding of human telomere G-quadruplex DNA probed by integrated fluorescence and magnetic tweezers spectroscopy. Nucleic Acids Res. 41, 2746 (2013).Ghimire, C. et al. Direct Quantification of Loop Interaction and pi-pi Stacking for G-Quadruplex Stability at the Submolecular Level. J. Am. Chem. Soc. 136, 15544 (2014).Garavís, M. et al. Mechanical Unfolding of Long Human Telomeric RNA (TERRA). Chem. Commun. 49, 6397 (2013).Fonseca Guerra, C., Zijlstra, H., Paragi, G. & Bickelhaupt, F. M. Telomere structure and stability: covalency in hydrogen bonds, not resonance assistance, causes cooperativity in guanine quartets. Chemistry-A European Journal 17, 12612 (2011).Yurenko, Y. P., Novotn, J., Sklen, V. & Marek, R. Exploring non-covalent interactions in guanine-and xanthine-based model DNA quadruplex structures: a comprehensive quantum chemical approach. Phys. Chem. Chem. Phys. 16, 2072 (2014).Poudel, L. et al. Implication of the solvent effect, metal ions and topology in the electronic structure and hydrogen bonding of human telomeric G-quadruplex DNA. Phys. Chem. Chem. Phys. 18, 21573 (2016).Li, M. H., Luo, Q., Xue, X. G. & Li, Z. S. Toward a full structural characterization of G-quadruplex DNA in aqueous solution: Molecular dynamics simulations of four G-quadruplex molecules. J. Mol. Struct-Theochem. 952, 96 (2010).Islam, B. et al. Conformational dynamics of the human propeller telomeric DNA quadruplex on a microsecond time scale. Nucleic Acids Res. 41, 2723 (2013).Stadlbauer, P., Krepl, M., Cheatham, T. E., Koca, J. & Sponer, J. Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations. Nucleic Acids Res. 41, 7128 (2013).Li, H., Cao, E. & Gisler, T. Force-induced unfolding of human telomeric G-quadruplex: a steered molecular dynamics simulation study. Biochem. Bioph. Res. Co. 379, 70 (2009).Yang, C., Jang, S. & Pak, Y. Multiple stepwise pattern for potential of mean force in unfolding the thrombin binding aptamer in complex with Sr2+. J. Chem. Phys. 135, 225104 (2011).Bergues-Pupo, A. E., Arias-Gonzalez, J. R., Morón, M. C., Fiasconaro, A. & Falo, F. Role of the central cations in the mechanical unfolding of DNA and RNA G-quadruplexes. Nucleic Acids Res. 43, 7638 (2015).Linak, M. C., Tourdot, R. & Dorfman, K. D. Moving beyond Watson-Crick models of coarse grained DNA dynamics. J. Chem Phys. 135, 205102 (2011).Rebi, M., Mocci, F., Laaksonen, A. & Ulin, J. Multiscale simulations of human telomeric G-quadruplex DNA. J. Phys. Chem. B 119, 105 (2014).Stadlbauer, P. et al. Coarse-Grained Simulations Complemented by Atomistic Molecular Dynamics Provide New Insights into Folding and Unfolding of Human Telomeric G-Quadruplexes. J. Chem. Theory Comput. 12, 6077 (2016).Parkinson, G. N., Lee, M. P. & Neidle, S. Crystal structure of parallel quadruplexes from human telomeric DNA. Nature 417, 876 (2002).Bhattacharya, D., Arachchilageand, G. M. & Basu, S. Metal Cations in G-Quadruplex Folding and Stability. Frontiers in Chemistry 4, 38 (2016).de Lorenzo, S., Ribezzi-Crivellari, M., Arias-Gonzalez, J. R., Smith, S. B. & Ritort, F. A Temperature-Jump Optical Trap for Single-Molecule Manipulation. Biophys. J. 108, 2854 (2015).Smith, S. B., Cui, Y. & Bustamante, C. Optical-trap force transducer that operates by direct measurement of light momentum. Methods Enzymol. 361, 134 (2003).Mergny, J. L., Phan, A. T. & Lacroix, L. Following G-quartet formation by UV-spectroscopy. FEBS letters 435, 74 (1998).Torrie, G. M. & Valleau, J. P. Nonphysical sampling distributions in Monte Carlo free-energy estimation: umbrella sampling. J. Comput. Phys. 23, 187 (1977).Kumar, S., Bouzida, D., Swendsen, R. H., Kollman, P. A. & Rosenberg, J. M. The weighted histogram analysis method for free-energy calculations on biomolecules I. The method. J. Comput. Chem. 13, 1011 (1992).Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72, 1541 (1997).Dudko, O. K., Hummer, G. & Szabo, A. Intrinsic rates and activation free energies from single-molecule pulling experiments. Phys. Rev. Lett. 96, 108101 (2006).Friddle, R. W., Noy, A. & De Yoreo, J. J. Interpreting the widespread nonlinear force spectra of intermolecular bonds. Proc. Natl. Acad. Sci. 109, 13573 (2012)

Thermal and inertial resonances in DNA unzipping

Single-molecule experiments combined with alternate forces are able to provide useful information not present in standard constant-force and -velocity pulling protocols. Here, we study the effects of such forces in the DNA mechanical unzipping by using an extension of the Peyrard-Bishop-Dauxois model. By changing the damping regime in the dynamical equations, we obtained two resonant mechanisms in both the mean time and the mean force of unzipping. One is thermally assisted and it is characterized by a matching between the period of the external force and the mean unzipping time of the DNA chain, while the other depends on the inertial properties of the system. Both mechanisms are studied systematically under different opening protocols and different parameters of the system. The main results here presented contribute in characterizing and finding optimized conditions in DNA unzipping experiments

Modelling the DNA topology: the effect of the loop bending on G-quadruplex stability

The guanine quadruplexes are DNA/RNA guanine-rich sequences assembled into four-stranded helical structures having important roles in gene regulation and chromosome stability. Their mechanical unfolding has been generally studied with all-atom simulations, which cannot dissect the specific interactions responsible for their cohesion. Recently, we introduced a mesoscopic model able to describe the main thermal and mechanical unfolding features of the G-quadruplex, with no distinction between the different geometrical conformations forming the structure. Under the same model we study here the contribution of the bending potential in the extra-structure bonding (loops). We demonstrate with numerical simulations that the rigidity of the loops can account for the different stabilities of different topologies, specifically the parallel and the antiparallel. In the presence of the bending, the model indicates the antiparallel configuration as the most stable G4 topology, as suggested by the experiments