Tuesday, February 14, 2017

DNA interaction with DAPI fluorescent dye: Force spectroscopy decouples two different binding modes

L. A. Reis, M. S. Rocha

In this work we use force spectroscopy in order to investigate the interaction between the DAPI fluorescent dye and the λ-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 (CT ∼ 0.50 μM) and presents equilibrium binding constants of the order of ∼ 107 M– 1 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 (∼ 105 M– 1).

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