Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T) 4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex

The binding of Hoechst 33258 and DAPI to five different (A/T) 4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K a = 5.5 × 10 8 M −1 for DAPI), and their affinities decrease in the series AATT ⪢ TAAT ≈ ATAT > TATA ≈ TTAA. The extreme values of K a differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T) 4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T) 4 sites are similar (2.46 × 10 8 M −1 s −1 to 0.86 × 10 8 M −1 s −1) and nearly diffusion-controlled, while the dissociation-rate parameters vary from 0.42 s −1 to 96 s −1. Thus the association constants are kinetically controlled and are close to their equilibrium-determined values. However, when obtained with increased signal-to-noise ratio, the kinetic traces for Hoechst 33258 binding at the AATT site reveal two components. The concentration dependencies of the two time constants and amplitudes are consistent with two different kinetically equivalent two-step models. In the first model, fast bimolecular binding is followed by an isomerization of the initial complex. In the second model, two single-step associations form two complexes that mutually exclude each other. For both models the four reaction-rate parameters are calculated. Finally, specific dissociation kinetics, using poly[d(A-5BrU)], show that the kinetics are even more complex than either two-step model. We correlate our results with the different binding orientations and locations of Hoechst 33258 in the DNA minor groove found in several structural studies in the literature.