title: Specific recognition of supercoiled plasmid DNA by affinity chromatography using the intercalator DAPP as ligand.
abstract: 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 10mM acetate buffer pH 5. Selective elution of oc and linear isoforms was achieved with 0.22M of sodium chloride in the same buffer. Finally, increasing the concentration to 0.55M 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.
authors.affiliation: CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Caramelo-Nunes
authors.forename: P
authors.initials: P
authors.lastname: Almeida
authors.forename: J C
authors.initials: JC
authors.lastname: Marcos
authors.forename: C T
authors.initials: CT
authors.lastname: Tomaz
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004278:DNA, Superhelical / Q000302:isolation & purification*; D004996:Ethidium / Q000031:analogs & derivatives; D007364:Intercalating Agents / Q000737:chemistry*; D008024:Ligands; D010617:Phenanthridines / Q000737:chemistry*; D010957:Plasmids / Q000302:isolation & purification*
chemical_list: D004278:DNA, Superhelical; D007364:Intercalating Agents; D008024:Ligands; D010617:Phenanthridines; C029154:3,8-diamino-6-phenyl-phenanthridine; D004996:Ethidium