Specific recognition of supercoiled plasmid DNA by affinity chromatography using the intercalator DAPP as ligand

• DAPP-Sepharose affinity chromatography.
• Separation of sc isoform from the less active oc and linear isoforms.
• The binding of pDNA to DAPP-Sepharose varies in function of pH.
• Protonated DAPP molecules bind more strongly to pDNA.

Small molecules that bind DNA with high specificity present a promising opportunity for application as chromatographic ligands for plasmid DNA (pDNA) purification. This research used the intercalator 3,8-diamino-6-phenylphenanthridine (DAPP) as an immobilized ligand for the specific separation of supercoiled (sc) pDNA by affinity chromatography. The results showed that the protonated DAPP-Sepharose support has a great affinity for sc pDNA isoform, separating it from the less active open circular and linear isoforms. All pDNA isoforms were retained in the column using 10 mM acetate buffer pH 5. Selective elution of oc and linear isoforms was achieved with 0.22 M of sodium chloride in the same buffer. Finally, increasing the concentration to 0.55 M led to the elution of the sc isoform. The binding of pDNA to DAPP-Sepharose varies in function of pH, and the stability of the protonated DAPP–DNA complex decreases with increasing salt concentration.