pDNA Cleavage Assay with ASRE

A pDNA cleavage assay with the use of pBR322 plasmid (New England BioLabs, Inc., N3033 L, Ipswich, MA, USA) was performed as reported previously [31 (link)]. In this assay, all samples contained 0.2 μg pDNA in the final sodium phosphate buffer (25 mmol L⁻¹ sodium phosphate, 50 μmol L⁻¹ DTPA, pH 7.4). Five microliters of the Na₂S, Na₂S_n, Cys or GSH aqueous stock solution (in final 100 μmol L⁻¹) was added to 15 µL of pDNA solution containing ASRE (in mg mL⁻¹: 0, 0.23, 0.47, 0.94, 1.41 or 1.88) or its chemical component (in mmol L⁻¹: 0.2, 0.4, 0.8, 1.2 or 1.6). All listed concentrations were final and calculated for a 20 µL sample. All ASRE components were prepared in water, except PA, which was dissolved in 100 mmol L⁻¹ sodium phosphate. The resulting samples 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 Image Studio analysis software (LI-COR Biotechnology, Bad Homburg, Germany) to estimate pDNA cleavage efficiency.