Correlation of Local Effects of DNA Sequence and Position of β-Alanine Inserts with Polyamide-DNA Complex Binding Affinities and Kinetics

To improve our understanding of the effects of beta-alanine (beta) substitution and the number of heterocycles on DNA binding affinity and selectivity, we investigated the interactions of an eight-ring hairpin polyamide (PA) and two beta derivatives as well as a six-heterocycle analogue with their cognate DNA sequence, 5'-TGGCTT-3'. Binding selectivity and the effects of beta have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV melting, biosensor surface plasmon resonance, isothermal titration calorimetry, circular dichroism, and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative Delta C-p and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that beta substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in the binding of PA to the mutant DNAs further confirm the position-dependent effects on the PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T.A versus A.T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of beta substitution on binding tell a clear and very important story about sequence-dependent binding of PAs to DNA.