Kinetic characterization of small DNA-binding molecules interacting with a DNA strand on a quartz crystal microbalance

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (k_on, k_off, and K_a, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA₃₀·dT₃₀ and dG₃₀·dC₃₀) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA₃₀·dT₃₀ and dG₃₀·dC₃₀, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG₃₀·dC₃₀ with high K_a and small k_off values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low k_on and k_off values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high K_a values owing to the high k_on values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.