Kinetic consequences of covalent linkage of DNA binding polyamides

Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subunits can bind in the minor groove of DNA at predetermined sequences with subnanomolar affinity and high specificity. Covalent linkage of polymer subunits using a gamma-aminobutyric acid linker has been shown to increase both the affinity and specificity of polyamides. Using a fluorescence detected stopped-flow assay, we have studied the differences in association and dissociation kinetics of a series of polyamides representing unlinked, hairpin and cyclic analogues of the four ring polyamide ImPyPyPy-beta-Dp. Whereas the large differences seen in the equilibrium association constants between the unlinked and covalently linked polyamides are primarily due to higher association rate constants, discrimination between matched and mismatched sites by each polyamide can be ascribed in large part to differences in their dissociation rate constants. The consequences of this kinetic behavior for future design are discussed.