DNA Cleavage Assay of Copper Complexes

The nuclease activity of the compounds was determined using plasmid pRS325II DNA, which was a gift from Steven Haase (Addgene plasmid #35467, 6835 base pairs) [71 (link)]. The plasmid was amplified in Escherichia coli by transforming One Shot^® TOP10 chemically competent E. coli (Invitrogen, Thermo Fisher Scientific). The plasmid was isolated from positive colonies using a PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen, Thermo Fisher Scientific). Plasmid (200 ng/sample) was exposed to complexes (1) and (2) in the presence of 1 mM hydrogen peroxide (to exploit the Cu(II/III) redox couple) or 1 mM ascorbic acid (to exploit the Cu(I/II) redox couple) [46 (link)]. The DNA cleavage experiments were done in a 9/1 (v/v) ratio of 50 mM Tris-HCl, pH 8, and DMSO. The samples were incubated for 1 h at 37 °C before a bromophenol blue/xylene cyanol-based loading dye (Roth, Germany) was added. The samples were loaded onto a 1% (w/v) agarose gel containing 1 μg/mL EtBr in 1 × TBE (Tris–boric acid–EDTA) buffer. Electrophoresis was performed at 50 V for 60 min in 1 × TBE buffer. The images of the fluorescent ethidium bromide-stained gels were captured using a gel documentation system (Doc-Print II, VilberLourmat, France). The cleavage experiments were done three times, with similar results. One representative gel is shown.