Micromethod for the investigation of the interactions between DNA and redox active molecules

A novel microscale and surface-based method for the study of the interactions of DNA with other redox-active molecules using DNA-modified electrodes is described. The method is simple, convenient, reliable, reagent-saving, and applicable for DNA studies, especially those involving microsamples, Information such as binding site size (s, in base pairs), binding constant (II), ratio (K-Ox/K-Red) of the binding constants for the oxidized and reduced forms of a bound species, binding free energy (Delta G(b)), and interaction mode, including changes in the mode of interaction, and "limiting" ratio K-Ox(degrees)/K-Red(degrees) at zero ionic strength can be obtained using only 3-15 mu g of DNA samples. The method was developed using [Co(Phen)(3)](3+/2+) (Phen = 1,10-phenanthroline)/double-stranded DNA (dsDNA)-modified gold electrodes and [Co(bpy)(3)](3+/2+) (2,2'-bipyridyl)/dSDNA-modified gold electrodes as model systems. For the [Co(Phen)(3)](3+/2+)/dsDNA-modified gold electrode system, a K2+ of (2.5 +/- 0.3) x 10(5) M-1 and an s of 5 bp were obtained in 5 mM pH 7.1 Tris-HCl buffer solution containing 50 min NaCl. For [Co(bpy)(3)](3+/2+)/dsDNA-modified gold electrodes, K3+ and s values of (1.3 +/- 0.3) x 10(5) M-1 and 3 bp, respectively, were obtained. While the s values are consistent with those reported in the literature obtained by solution methods, the IZ values are almost an order of magnitude larger. A transition in the nature of the interaction between dsDNA and [Co(Phen)(3)](3+/2+), from electrostatic to intercalative with increasing ionic strength, was found in our studies. Negative values of Delta E degrees' for [Co(bpy)(3)](3+/2+) bound to dsDNA suggest that its interaction with dsDNA is predominantly electrostatic over the ionic strength range of 5-105 mM. The "limiting" ratio K(3+)degrees/K(2+)degrees of 22 obtained for [Co(Phen)(3)](3+/2+) bound to dsDNA at zero ionic strength suggests that electrostatic interactions are predominant over intercalative ones under these limiting conditions. The ratio for [Co(bpy)(3)](3+/2+) of 16 also indicates that the 3+ form binds to dsDNA more strongly than the 2+ form at zero ionic strength. For [Co(Phen)(3)](3+/2+)/single-stranded DNA (ssDNA)-modified gold electrodes, the nonuniformity of the surface structure of ssDNA-modified gold electrodes greatly complicates the analysis. A system consisting of a dsDNA-modified gold electrode and [Co(tppz)(2)](3+/2+) (tppz = tetra-2-pyridyl-1,4-pyrazine) was studied by this method, with a K2+ value of (5 +/- 1) x 10(5) M-1 and an s value of 7 bp being obtained.