Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11797
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Resultados da Pesquisa
Item Carboplatin as an alternative to Cisplatin in chemotherapies: New insights at single molecule level(Biophysical Chemistry, 2018-10) Oliveira, L.; Caquito Jr., J. M.; Rocha, M. S.Here we report a new study performed at single molecule level on the interaction of the antineoplastic drug Carboplatin and the DNA molecule - the main target of the drug inside cells in cancer chemotherapies. By using optical tweezers, we measure how the mechanical properties of the DNA-Carboplatin complexes changes as a function of the drug concentration in the sample, for two different ionic strengths ([Na] = 150 mM and [Na] = 1 mM). From these measurements, the binding mechanism and the physicochemical (binding) parameters of the interaction were inferred and directly compared to those obtained for the precursor drug Cisplatin under equivalent conditions. As the main conclusion, we show that Carboplatin binds preferentially forming covalent monoadducts in contrast to Cisplatin, which is hydrolyzed easier and presents a higher efficiency in forming covalent diadducts along the double-helix. In addition, we explicitly show that Carboplatin is much less sensitive to ionic strength changes when compared to Cisplatin. These findings provide new insights on the interactions of platinum-based compounds with the DNA molecule, being important to improve the current treatments and in the development of new antineoplastic agents.Item Videomicroscopy calibration of optical tweezers by position autocorrelation function analysis(Applied Physics B, 2012-03-21) Alves, P. S.; Rocha, M. S.We present a simple method to calibrate optical tweezers by using only videomicroscopy to measure the position autocorrelation function of the trapped bead in the potential well of the tweezers. To accomplish this task, we use a high-speed camera with ∼500 fps (frames per second), which provides a precise measurement of the relaxation time of the bead Brownian fluctuations. We also study the variation of the trap stiffness as a function of some parameters of interest such as the laser power and the distance from the bead center to the microscope coverslip, showing that the presented method returns precise results.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 Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments(Integrative Biology, 2015-08-06) Rocha, M. S.In this review we focus on the idea of establishing connections between the mechanical properties of DNA–ligand complexes and the physical chemistry of DNA–ligand interactions. This type of connection is interesting because it opens the possibility of performing a robust characterization of such interactions by using only one experimental technique: single molecule stretching. Furthermore, it also opens new possibilities in comparing results obtained by very different approaches, in particular when comparing single molecule techniques to ensemble-averaging techniques. We start the manuscript reviewing important concepts of DNA mechanics, from the basic mechanical properties to the Worm-Like Chain model. Next we review the basic concepts of the physical chemistry of DNA–ligand interactions, revisiting the most important models used to analyze the binding data and discussing their binding isotherms. Then, we discuss the basic features of the single molecule techniques most used to stretch DNA–ligand complexes and to obtain “force × extension” data, from which the mechanical properties of the complexes can be determined. We also discuss the characteristics of the main types of interactions that can occur between DNA and ligands, from covalent binding to simple electrostatic driven interactions. Finally, we present a historical survey of the attempts to connect mechanics to physical chemistry for DNA–ligand systems, emphasizing a recently developed fitting approach useful to connect the persistence length of DNA–ligand complexes to the physicochemical properties of the interaction. Such an approach in principle can be used for any type of ligand, from drugs to proteins, even if multiple binding modes are present.Item Controlling cooperativity in β-cyclodextrin–DNA binding reactions(The Journal of Physical Chemistry Letters, 2015-08-26) Rocha, M. S.; Alves, P. S.; Mesquita, O. N.We have investigated the interaction between the native neutral β- cyclodextrin (CD) and the DNA molecule by performing single-molecule stretching experiments with optical tweezers. In particular, we have monitored the changes of the mechanical properties of the CD−DNA complexes as a function of the CD concentration in the sample. By using a quenched disorder statistical model, we were also capable to extract important physicochemical information (equilibrium binding constants, cooperativity degree) of such interaction from the mechanical data. In addition, we have found that the interaction occurs by two different mechanisms, first with the formation of relatively large CD clusters along the double helix, which thereafter can locally denature the DNA molecule by forming hydrogen bonds with the base pairs that eventually flip out. A prediction of our quenched disorder model was that cooperativity could be controlled by adjusting the surface charge of β-CD molecules. This prediction is confirmed in the present work.Item Force spectroscopy unravels the role of ionic strength on DNA-cisplatin interaction: Modulating the binding parameters(Physical Review E, 2017-09-11) Oliveira, L.; Rocha, M. S.In the present work we have gone a step forward in the understanding of the DNA-cisplatin interaction, investigating the role of the ionic strength on the complexes formation. To achieve this task, we use optical tweezers to perform force spectroscopy on the DNA-cisplatin complexes, determining their mechanical parameters as a function of the drug concentration in the sample for three different buffers. From such measurements, we determine the binding parameters and study their behavior as a function of the ionic strength. The equilibrium binding constant decreases with the counterion concentration ([Na]) and can be used to estimate the effective net charge of cisplatin in solution. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength, as a result of the different conformational changes induced by the drug on the double-helix when binding under different buffer conditions. Such results can be used to modulate the drug binding to DNA, by appropriately setting the ionic strength of the surrounding buffer. The conclusions drawn provide significant new insights on the complex cooperative interactions between the DNA molecule and the class of platinum-based compounds, much used in chemotherapies.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.