Electrochemical Determination of Nucleic Acid Diffusion Coefficients through Noncovalent Association of a Redox-Active Probe

Abstract

The diffusion coefficients of sonicated calf-thymus DNA and an oligonucleotide fragment have been measured via an electrochemical strategy. The complex [Os(bpy)2(dppz)12+ (bpy = 2, 2'-bipyridine, dppz = dipyridophenazine) has been synthesized as a simple, single-electron intercalating redox couple. The free complex undergoes a kinetically reversible single-electron oxidation to the corresponding Os(II1) complex at an applied potential of 0.72 V vs Ag/AgCl, which is insufficient to achieve electrocatalytic DNA oxidation.Upon addition of DNA, the complex binds via intercalation to a two-base-pair site with binding constant Kb = 4 x lo6 M-I, as reflected in the changes in the photophysics of the Os(I1)-dppz chromophore. Changes in the voltammetric current upon binding reveal a slight limitation on heterogeneous charge-transfer kinetics (k = 5 x lov4 cm s-I) and a dramatic limitation on mass transfer of bound complexes. Normal pulse voltammetry gives diffusion coefficients of sonicated calf-thymus DNA and (dA)20*(dT)20 equal to 2.0 x cm2 s-I, respectively; each agrees with values reported for light-scattering experiments and theoretical calculation. and 1.2 xThe diffusion properties of biopolymers play an important role in many physiological For example, the recognition of a specific cleavage site by restriction enzymes is thought to involve initial, nonspecific electrostatic binding of the enzyme to DNA in a mode that is weak enough that diffusion of the enzyme is still f a~i l e .~ The weakly bound enzyme then diffuses along the DNA strand until reaching the cleavage locus and effecting site-specific cleavage. The importance of these processes clearly dictates a detailed understanding of the relative diffusion properties of molecules such as DNA and DNA-binding proteins. We report here on an electrochemical system for studying the diffusion properties of nucleic acids that can be performed on small volumes of sample at relatively low nucleic acid concentrations. These studies can be performed with commercially available potentiostats and analysis software.The discovery of both natural transition-metal-based antitumor agents such as iron-bleomycin (FeBLM)4*5 and synthetic platinum drugs6 has prompted intense investigation of the interactions and reactions of synthetic transition-metal complexes with nucleic acids. In particular, extensive efforts have been made to cleave DNA oxidatively in a site-specific manner with metal complexes in either an electronic excited or electrochemically potentiated high oxidation state.I0,' Electrochemical studies have been made of electrocatalytic cleavage reactions using FeBLM,' Mn porphyrins,I0 outer-sphere oxidants,I2 and complexes based on the Ru(IV)O~+/RU(II)OH~~+ ~o u p l e . '~-'~ An advantage of electrochemical cleavage reactions is that the current measured in electrolysis or in voltammetry should provide mechanistic information on the extent or rate of the cleavage reaction. However, because currents in solution electrochemistry are governed by mass transport, reliable inte...

DNA interaction with dipolar ruthenium(II) ammine complexes containing 4,4′-bipyridinium as photochemotherapeutic agents

“…The observed shift to a more negative potential in E 1/2 for Ru(III/ II) couple (DE°0 = À20 mV at r = 5) is similar to that of related metal(III/II) couples [47][48][49][50], suggesting preferential stabilization of the …”

Determination of Surface-Immobilized Double-Stranded DNA Using a Metallointercalator and Catalytic Voltammetry

“…Firstly, its oxidation potential is about 1.25 V vs. Ag=AgCl, and it is able to oxidize oxalate with high efficiency. Secondly, similar ruthenium and osmium complexes with dppz ligands have been shown in fluorescence and electrochemistry studies to intercalate into ds-DNA with relatively high affinity (K ¼ 10 6 -10 7 M À1 ) and selectivity [37][38][39][40]. Figure 5 illustrates the binding selectivity of Ru-dppz toward ds-DNA films assembled on an electrode surface.…”

Synthesis, characterization and DNA-binding properties of rac-[Ru(5,6-dmp)2(dppz)]2+ – Enantiopreferential DNA binding and co-ligand promoted exciton coupling

“…The formal potential of the Ru(II)/Ru(III) couple E 00 (or voltammetric E 1/2 ) for the 5,6-dmp complex (1.189 V) is lower than the phen analogue (1.201 V) revealing that the incorporation of the 5,6-dimethyl groups on the phen ring renders the oxidation of Ru(II) to Ru(III) more facile. On the incremental addition of DNA to the complexes (R = 0.5-4) the oxidation peak currents decrease indicating the strong DNA binding of both the complexes [68] (Fig. 10(a) and (b)) and beyond R = 3 the DPV responses are flattened.…”