Kinetic consequences of covalent linkage of DNA binding polyamides

Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subu nits can bind in the minor groove of DNA at predetermined sequences with su bnanomolar affinity and high specificity. Covalent linkage of polymer subun its 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 diss ociation 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 s ites by each polyamide can be ascribed in large part to differences in thei r dissociation rate constants. The consequences of this kinetic behavior fo r future design are discussed.