Abstract A series of three-ring analogs of the minor-groove-binding molecule Hoechst 33258 (1), consisting of benzimidazole (B), imidazopyridine (P), and hydroxybenzimidazole (H) monomers, have been synthesized in order to investigate both their sequence specificity and binding modes. MPE⋅ Fe II Footprinting has revealed the preference of both PBB and BBB ligands for 5′-WGWWW-3′ and 5′-WCWWW-3′ tracts, as well as A⋅ T-rich ...