pDNA Cleavage Assay with pBR322 Plasmid

pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs Inc., N3033L) was performed as reported previously [5 (link), 36 (link)]. In this assay, all samples contained 0.2 μg pDNA in sodium phosphate buffer (25 mmol L⁻¹ sodium phosphate, 50 μmol L⁻¹ DTPA, pH 7.4, 37°C). After addition of compounds, the resulting mixtures were incubated for 30 min at 37°C. All concentrations listed in the section were final in the samples. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Samples were electrophoresed in TBE buffer (89 mmol L⁻¹ Tris, 89 mmol L⁻¹ boric acid, and 2 mmol L⁻¹ EDTA) at 5.5 V cm⁻¹ for 2 h; gels were stained with GelRed™ Nucleic Acid Gel Stain and photographed using a UV transilluminator. Integrated densities of all pBR322 forms in each lane were quantified using the TotalLab TL100 image analysis software to estimate pDNA cleavage efficiency (Nonlinear Dynamic Ltd., USA).