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 arra ngements of AT residues, using synthetic DNA fragments containing mult iple blocks of (A/T)(6) or (A/T)(10) in identical sequence environment s, 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 dis tamycin shows little discrimination between the various sites, binding similar to 2-fold stronger to TAATTA than (TA)(3), T(3)A(3) and GAATT C. 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 footpri nting reveals that both ligands bind in similar locations at the centr e of each AT tract, At (A/T)10 sites distamycin binds with similar aff inity 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 thos e towards the edges, Hoechst 33258 shows a similar pattern of preferen ce, with strong binding to AATT, T(5)A(5) and the centre of (TA)(5). H ydroxyl radical footprinting reveals that at low concentrations both l igands bind at the centre of (TA)(5) and A(5)T(5), while at higher con centrations 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 th at these compounds avoid the TpA step, Similar DNase I footprinting ex periments 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 .