Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11797
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Resultados da Pesquisa
Item DNA interaction with hoechst 33258: stretching experiments decouple the different binding modes(The Journal of Physical Chemistry B, 2013-05-28) Silva, E. F.; Ramos, E. B.; Rocha, M. S.By performing single molecule stretching experiments with optical tweezers, we have studied the DNA interaction with the ligand Hoechst 33258. The mechanical properties of the complexes formed as a function of ligand concentration were directly determined from these measurements by fitting the force × extension curve to the WormLike Chain model of semiflexible polymers. In addition, the physicochemical parameters of the interaction were extracted from the persistence length data by using a previously developed two-sites quenched disorder statistical model, allowing the determination of the binding isotherm. Such approach has allowed us to decouple the two different binding modes present in this system. In particular, it was found that the binding isotherm consists of two Hill-type processes, one noncooperative and the other strongly cooperative. Finally, DNA condensation due to the interaction with the ligand was also verified and characterized here by analyzing the apparent contour length of the complexes.Item Force-dependent persistence length of DNA–intercalator complexes measured in single molecule stretching experiments(Soft Matter, 2015-04-20) Bazoni, R. F.; Lima, C. H. M.; Ramos, E. B.; Rocha, M. S.By using optical tweezers with an adjustable trap stiffness, we have performed systematic single molecule stretching experiments with two types of DNA–intercalator complexes, in order to investigate the effects of the maximum applied forces on the mechanical response of such complexes. We have explicitly shown that even in the low-force entropic regime the persistence length of the DNA–intercalator complexes is strongly force-dependent, although such behavior is not exhibited by bare DNA molecules. We discuss the possible physicochemical effects that can lead to such results. In particular, we propose that the stretching force can promote partial denaturation on the highly distorted double-helix of the DNA–intercalator complexes, which interfere strongly in the measured values of the persistence length.Item On the effects of intercalators in DNA condensation: a force spectroscopy and gel electrophoresis study(The Journal of Physical Chemistry B, 2014-04-10) Rocha, M. S.; Cavalcante, A. G.; Silva, R.; Ramos, E. B.In this work we have characterized the effects of the intercalator ethidium bromide (EtBr) on the DNA condensation process by using force spectroscopy and gel electrophoresis. We have tested two condensing agents: spermine (spm4+), a tetravalent cationic amine which promotes cation-induced DNA condensation, and poly(ethylene glycol) (PEG), a neutral polymer which promotes DNA ψ-condensation. Two different types of experiments were performed. In the first type, bare DNA molecules disperse in solution are first treated with EtBr for intercalation, and then the condensing agent is added to the sample with the purpose of verifying the effects of the intercalator in hindering DNA condensation. In the second experiment type, the bare DNA molecules are first condensed, and then the intercalator is added to the sample in order to verify its influence on the previously condensed DNA. The results obtained with the two different experimental techniques used agree very well, indicating that previously intercalated EtBr can hinder both cation-induced and ψ-condensation, being more efficient in the first case. On the other hand, EtBr has little effect on the previously formed cation-induced condensates, but is efficient in unfolding the ψ-condensates.Item DNA Interaction with diaminobenzidine studied with optical tweezers and dynamic light scattering(The Journal of Physical Chemistry B, 2013-10-28) Reis, L. A.; Ramos, E. B.; Rocha, M. S.We have studied the interaction of the DNA molecule with the ligand 3,3′-diaminobenzidine (DAB) by performing single molecule stretching experiments with optical tweezers and dynamic light scattering (DLS) on the DNA−DAB complexes. In the stretching experiments, the persistence and contour lengths of the complexes were measured as a function of DAB concentration, allowing one to infer the main binding mechanism and also to determine the physicochemical parameters of the interaction. In the DLS experiments, the effective size of the complexes, measured as the hydrodynamic radius, was monitored as a function of DAB concentration. We found a qualitative agreement between the results obtained from the two techniques by comparing the behaviors of the hydrodynamics radius and the radius of gyration, since this last one can be expressed as a function of the persistence and contour lengths.Item Depletion interactions and modulation of DNA‐intercalators binding: Opposite behavior of the “neutral” polymer poly(ethylene‐glycol)(Biopolymers, 2015-11-30) Crisafuli, F. A. P.; Silva, L. H. M. da; Ferreira, G. M. D.; Ramos, E. B.; Rocha, M. S.; Walter, NilsIn this work we have investigated the role of high molecular weight poly(ethylene‐glycol) 8000 (PEG 8000) in modulating the interactions of the DNA molecule with two hydrophobic compounds: Ethidium Bromide (EtBr) and GelRed (GR). Both compounds are DNA intercalators and are used here to mimic the behavior of more complex DNA ligands such as chemotherapeutic drugs and proteins whose domains intercalate DNA. By means of single‐molecule stretching experiments, we have been able to show that PEG 8000 strongly shifts the binding equilibrium between the intercalators and the DNA even at very low concentrations (1% in mass). Additionally, microcalorimetry experiments were performed to estimate the strength of the interaction between PEG and the DNA ligands. Our results suggest that PEG, depending on the system under study, may act as an “inert polymer” with no enthalpic contribution in some processes but, on the other hand, it may as well be an active (non‐neutral) osmolyte in the context of modulating the activity of the reactants and products involved in DNA‐ligand interactions.Item Unfolding DNA condensates produced by DNA-like charged depletants: A force spectroscopy study(The Journal of Chemical Physics, 2017-02-02) Lima, C. H. M.; Rocha, M. S.; Ramos, E. B.In this work, we have measured, by means of optical tweezers, forces acting on depletion-induced DNA condensates due to the presence of the DNA-like charged protein bovine serum albumin (BSA). The stretching and unfolding measurements performed on the semi-flexible DNA chain reveal (1) the softening of the uncondensed DNA contour length and (2) a mechanical behavior strikingly different from those previously observed: the force-extension curves of BSA-induced DNA condensates lack the “saw-tooth” pattern and applied external forces as high as ≈80 pN≈80 pN are unable to fully unfold the condensed DNA contour length. This last mechanical experimental finding is in agreement with force-induced “unpacking” detailed Langevin dynamics simulations recently performed by Cortini et al. on model rod-like shaped condensates. Furthermore, a simple thermodynamics analysis of the unfolding process has enabled us to estimate the free energy involved in the DNA condensation: the estimated depletion-induced interactions vary linearly with both the condensed DNA contour length and the BSA concentration, in agreement with the analytical and numerical analysis performed on model DNA condensates. We hope that future additional experiments can decide whether the rod-like morphology is the actual one we are dealing with (e.g. pulling experiments coupled with super-resolution fluorescence microscopy).Item DNA interaction with Actinomycin D: mechanical measurements reveal the details of the binding data(Physical Chemistry Chemical Physics, 2013-05-07) Cesconetto, E. C.; A. Junior, F. S.; Crisafuli, F. A. P.; Mesquita, O. N.; Ramos, E. B.; Rocha, M. S.We have studied the interaction between the anticancer drug Actinomycin D (ActD) and the DNA molecule by performing single molecule stretching experiments and atomic force microscopy (AFM) imaging. From the stretching experiments, we determine how the mechanical properties of the DNA–ActD complexes vary as a function of drug concentration, for a fixed DNA concentration. We have found that the persistence lengths of the complexes formed behave non-monotonically: at low concentrations of ActD they are more flexible than the bare DNA molecule and become stiffer at higher concentrations. On the other hand, the contour length increases monotonically as a function of ActD concentration. Using a two-sites quenched disorder statistical model recently developed by us, we were able to extract chemical parameters such as the intrinsic binding constant and the degree of cooperativity from these pure mechanical measurements, thus performing a robust characterization of the interaction. The AFM images, otherwise, were used to measure the bending angle size distribution that ActD introduces on the double-helix structure and the average number of bendings per DNA molecule as a function of drug concentration, two quantities that cannot be determined from the stretching experiments.Item DNA-doxorubicin interaction: New insights and peculiarities(Biopolymers, 2016-10-05) Silva, E. F.; Bazoni, R. F.; Ramos, E. B.; Rocha, M. S.We have investigated the interaction of the DNA molecule with the anticancer drug doxorubicin (doxo) by using three different experimental techniques: single molecule stretching, single molecule imaging, and dynamic light scattering. Such techniques allowed us to get new insights on the mechanical behavior of the DNA-doxo complexes as well as on the physical chemistry of the interaction. First, the contour length data obtained from single molecule stretching were used to extract the physicochemical parameters of the DNA-doxo interaction under different buffer conditions. This analysis has proven that the physical chemistry of such interaction can be modulated by changing the ionic strength of the surrounding buffer. In particular we have found that at low ionc strengths doxo interacts with DNA by simple intercalation (no aggregation) and/or by forming bound dimers. For high ionic strengths, otherwise, doxo-doxo self-association is enhanced, giving rise to the formation of bound doxo aggregates composed by 3 to 4 molecules along the double-helix. On the other hand, the results obtained for the persistence length of the DNA-doxo complexes is strongly force-dependent, presenting different behaviors when measured with stretching or non-stretching techniques.