Interaction of minor groove binding ligands with long AT tracts

We have used quantitative DNase I footprinting to examine the ability of distamycin and Hoechst 33258 to discriminate between different arrangements of AT residues, using synthetic DNA fragments containing multiple blocks of (A/T)(6) or (A/T)(10) in identical sequence environments, Previous studies have shown that these ligands bind less well to (A/T)(4) sites containing TpA steps, We find that in (A/T)(6) tracts distamycin shows little discrimination between the various sites, binding similar to 2-fold stronger to TAATTA than (TA)(3), T(3)A(3) and GAATTC. In contrast, Hoechst 33258 binds similar to 20-fold more tightly to GAATTC and TAATTA than T(3)A(3) and (TA)(3). Hydroxyl radical footprinting reveals that both ligands bind in similar locations at the centre of each AT tract, At (A/T)10 sites distamycin binds with similar affinity to T(5)A(5), (TA)(5) and AATT, though bands in the centre of (TA)(5) are protected at similar to 50-fold lower concentration than those towards the edges, Hoechst 33258 shows a similar pattern of preference, with strong binding to AATT, T(5)A(5) and the centre of (TA)(5). Hydroxyl radical footprinting reveals that at low concentrations both ligands bind at the centre of (TA)(5) and A(5)T(5), while at higher concentrations ligand molecules bind to each end of the (A/T)(10) tracts, At T(5)A(5) two ligand molecules bind at either end of the site, even at the lowest ligand concentration, consistent with the suggestion that these compounds avoid the TpA step, Similar DNase I footprinting experiments with a DNA fragment containing T-n (n = 3-6) tracts reveals that both ligands bind in the order T-3 < T-4 much less than T-5 = T-6.