Centro de Ciências Exatas e Tecnológicas
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/9791
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Resultados da Pesquisa
Item Effects of caffeine on the structure and conformation of DNA: A force spectroscopy study(International Journal of Biological Macromolecules, 2019-06-01) Moura, T. A.; Oliveira, L.; Rocha, M.S.Here, we use single molecule force spectroscopy performed with optical tweezers in order to investigate the interaction between Caffeine and the DNA molecule for various different concentrations of the alkaloid and under two distinct ionic strengths of the surrounding buffer. We were able to determine the mechanical changes induced on the double-helix structure due to Caffeine binding, the binding mode and the binding parameters of the interaction. The results obtained show that Caffeine binds to DNA by outside the double-helix with a higher affinity at lower ionic strengths. On the other hand, a considerable cooperativity was found only for sufficient high ionic strengths, suggesting that Caffeine may binding forming dimers and/or trimers along the double-helix under this condition. Finally, it was also shown that Caffeine stabilizes the DNA double-helix upon binding, preventing force-induced DNA melting.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 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 Biophysical characterization of the DNA interaction with the biogenic polyamine putrescine: a single molecule study(International Journal of Biological Macromolecules, 2018-02-26) Publio, B.C.; Moura, T.A.; Lima, C.H.M.; Rocha, M.S.We have performed a biophysical characterization, at single molecule level, of the interaction between the DNA molecule and the biogenic polyamine putrescine. By using force spectroscopy, we were able to monitor the complexes formation as putrescine is added to the sample, determining the mechanical properties of such complexes and the physicochemical (binding) parameters of the interaction for three different ionic strengths. In particular, it was shown that the behavior of the equilibrium binding constant as a function of the counterion concentration deviates from the prediction of the Record-Lohman model. The measured constants were (1.3 ± 0.2) × 10^5 M^- 1 for [Na] = 150 mM, (2.1 ± 0.2) × 10^5 M^- 1 for [Na] = 10 mM, and (2.2 ± 0.3) × 10^5 M^- 1 for [Na] = 1 mM. The cooperativity degree of the binding reaction, on the other hand, increases with the ionic strength. From these analysis, the DNA-putrescine binding mechanisms are inferred, and a comparison with results reported for ordinary bivalent ions like magnesium is performed. Such study provides new insights on the general behavior of the DNA interactions with biogenic polyamines.Item Force and scale dependence of the elasticity of self-assembled DNA bottle brushes(Macromolecules, 2017-12-28) Rocha, Marcio Santos; Storm, Ingeborg M.; Bazoni, Raniella Falchetto; Ramos, Ésio Bessa; Garcia, Armando Hernandez; Stuart, Martien A. Cohen; Leermakers, Frans; Vries, Renko deAs a model system to study the elasticity of bottle-brush polymers, we here introduce self-assembled DNA bottle brushes, consisting of a DNA main chain that can be very long and still of precisely defined length, and precisely monodisperse polypeptide side chains that are physically bound to the DNA main chains. Polypeptide side chains have a diblock architecture, where one block is a small archaeal nucleoid protein Sso7d that strongly binds to DNA. The other block is a net neutral, hydrophilic random coil polypeptide with a length of exactly 798 amino acids. Light scattering shows that for saturated brushes the grafting density is one side chain per 5.6 nm of DNA main chain. According to small-angle X-ray scattering, the brush diameter is D = 17 nm. By analyzing configurations of adsorbed DNA bottle brushes using AFM, we find that the effective persistence of the saturated DNA bottle brushes is Peff = 95 nm, but from force−extension curves of single DNA bottle brushes measured using optical tweezers we find Peff = 15 nm. The latter is equal to the value expected for DNA coated by the Sso7d binding block alone. The apparent discrepancy between the two measurements is rationalized in terms of the scale dependence of the bottle-brush elasticity using theory previously developed to analyze the scale-dependent electrostatic stiffening of DNA at low ionic strengths.Item DNA interaction with DAPI fluorescent dye: Force spectroscopy decouples two different binding modes(Biopolymers, 2017-01-18) Reis, L. A.; Rocha, M. S.In this work, we use force spectroscopy to investigate the interaction between the DAPI fluorescent dye and the k-DNA molecule under high (174 mM) and low (34 mM) ionic strengths. Firstly, we have measured the changes on the mechanical properties (persistence and contour lengths) of the DNA-DAPI complexes as a function of the dye concentration in the sample. Then, we use recently developed models in order to connect the behavior of both mechanical properties to the physical chemistry of the interaction. Such analysis has allowed us to identify and to decouple two main binding modes, determining the relevant physicochemical (binding) parameters for each of these modes: minor groove binding, which saturates at very low DAPI concentrations (C T 0.50 lM) and presents equilibrium binding constants of the order of 10 7 M 21 for the two ionic strengths studied; and intercalation, which starts to play a significant role only after the saturation of the first mode, presenting much smaller equilibrium binding constants ( 10 5 M 21 ).