Centro de Ciências Exatas e Tecnológicas
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/9791
Navegar
5 resultados
Resultados da Pesquisa
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 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 ).