Cation binding to DNA studied by ion-transfer voltammetry at micropipets

Two new approaches to the study of ion binding to DNA have been developed. Both are based on measurement of ion transfer across the interface between two immiscible electrolyte solutions. In the first method, the cation of interest is initially present in the aqueous phase and transferred to the organic phase contained in a micropipet when its potential is made sufficiently;negative. Upon addition of high-molecular-weight DNA to the aqueous phase, the concentration of free cation decreases, which results in a decrease in the ion-transfer current. The corresponding binding constant can be extracted from the dependence of normalized steady-state current vs DNA concentration without the knowledge of micropipet size, binding kinetics, or diffusion coefficient values. In the second method, the cation of interest is present in the organic phase inside the pipet and oligonucleotides (fragments of DNA) are added to the external aqueous phase. The transfer of the cation to the aqueous phase may be facilitated by the oligonucleotides present in the aqueous phase. The facilitated transfer appears as a steady-state wave dependent on the concentration of oligonucleotides in the aqueous phase. The binding constant can be estimated from the shift in the transfer potential between the facilitated and nonfacilitated transfer. The cation chosen, N-methylphenanthroline, is a known DNA intercalator, and analysis of the steady-state wave for facilitated transfer allows an estimate (2.8) of the number of ions transferred per molecule of oligonucleotide arriving at the interface. The DNA-methylphenanthroline complex adsorbs at the interface, and a stripping peak for ex-traction of N-methylphenanthroline from the adsorbed DNA back into the organic phase is observed on reversing the scan direction.