We report the synthesis and characterization of photonucleases N,N'-bis[2-[bis(1H-imidazol-4-ylmethyl)amino]ethyl]3,6-acridinediamine (7) and N-[2-[bis(1H-imidazol-4-ylmethyl)amino]ethyl]-3,6-acridinediamine (10), consisting of a central 3,6-acridinediamine chromophore attached to 4 and 2 metal-coordinating imidazole rings, respectively. In DNA reactions employing 16 metal salts, photocleavage of pUC19 plasmid is markedly enhanced when compound 7 is irradiated in the presence of either Hg(II), Fe(III), Cd(II), Zn(II), V(V), or Pb(II) (low-intensity visible light, pH 7.0, 22 degrees C, 8-50,mu M 7). We also show that DNA photocleavage by 7 can be modulated by modifying buffer type and pH. Evidence of metal complex formation is provided by EDTA experiments and by NMR and electrospray ionization mass spectral data. Sodium azide, sodium benzoate, superoxide dismutase, and catalase indicate the involvement of type I and 11 photochemical processes in the metal-assisted DNA photocleavage reactions. Thermal melting studies show that compound 7 increases the T-m of calf thymus DNA by 10 1 degrees C at pH 7.0 and that the Tm is further increased upon the addition of either Hg(II), Cd(II), Zn(II), or Pb(II). In the case of Fe(III) and V(V), a colorimetric assay demonstrates that compound 7 sensitizes one electron photoreduction of these metals to Fe(II) and V(IV), likely accelerating the production of type I reactive oxygen species. Our data collectively indicate that buffer, pH, Hg(II), Fe(III), Cd(II), Zn(II), V(V), Pb(II), and light can be used to "tune" DNA cleavage by compound 7 under physiologically relevant conditions. The 3,6-acridinediamine acridine orange has demonstrated great promise for use as a photosensitizer in photodynamic therapy. In view of the distribution of iron in living cells, compound 7 and other metal-binding acridine-based photonucleases should be expected to demonstrate excellent photodynamic action in vivo.