Plasmid DNA Cleavage Assay

A pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs, Inc., N3033 L, Ipswich, MA, USA) was performed as reported previously [13 (link)]. In this assay, all samples contained 0.2 μg pDNA in the sodium phosphate buffer (25 mmol L⁻¹ sodium phosphate, 50 μmol L⁻¹ DTPA, pH 7.4). FeCl₂ (150 µmol L⁻¹) was used in control experiments. Powdered KO₂ was dissolved by 4 mmol L⁻¹ BMPO in the buffer containing 10% DMSO (v/v), vortexed for 10 s and 10 µL of the mixture was added into 10 µL solution of pDNA. The resulting mixtures were incubated for 30 min at 37 °C. After incubation, the reaction mixtures were subjected to 0.6% agarose gel electrophoresis. Integrated densities of all pBR322 forms in each lane were quantified using the Image Studio analysis software (LI-COR Biotechnology, Bad Homburg, Germany) to estimate pDNA-cleavage efficiency.