CATION-BINDING TO DNA STUDIED BY ION-TRANSFER VOLTAMMETRY AT MICROPIPETS

Two new approaches to the study of ion binding to DNA have been develo ped. Both are based on measurement of ion transfer across the interfac e between two immiscible electrolyte solutions. In the first method, t he cation of interest is initially present in the aqueous phase and tr ansferred to the organic phase contained in a micropipet when its pote ntial is made sufficiently;negative. Upon addition of high-molecular-w eight DNA to the aqueous phase, the concentration of free cation decre ases, which results in a decrease in the ion-transfer current. The cor responding binding constant can be extracted from the dependence of no rmalized steady-state current vs DNA concentration without the knowled ge of micropipet size, binding kinetics, or diffusion coefficient valu es. In the second method, the cation of interest is present in the org anic phase inside the pipet and oligonucleotides (fragments of DNA) ar e added to the external aqueous phase. The transfer of the cation to t he 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 aqueou s phase. The binding constant can be estimated from the shift in the t ransfer potential between the facilitated and nonfacilitated transfer. The cation chosen, N-methylphenanthroline, is a known DNA intercalato r, and analysis of the steady-state wave for facilitated transfer allo ws an estimate (2.8) of the number of ions transferred per molecule of oligonucleotide arriving at the interface. The DNA-methylphenanthroli ne complex adsorbs at the interface, and a stripping peak for ex-tract ion of N-methylphenanthroline from the adsorbed DNA back into the orga nic phase is observed on reversing the scan direction.