Specific berenil–DNA interactions: An approach for separation of plasmid isoforms by pseudo-affinity chromatography

Abstract

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6M and then to 0M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography.

Chemotherapeutic Drug‐Conjugated Microbeads Demonstrate Preferential Binding to Methylated Plasmid DNA

“…Berenil is an anti‐infective drug containing an aromatic diamidine structure that preferentially binds to DNA at the AATT sequence and has been previously employed for pDNA purification. Doxorubicin (adriamycin) is an anticancer drug which interacts with genomic DNA, ultimately preventing DNA replication . We conjugated doxorubicin and berenil (Figure S1a, Supporting Information) onto Amikabeads and investigated the efficacy of the resulting ligand‐conjugated microbeads for binding pDNA processed using E. coli that either possess or lack DNA methylation enzymes that methylate DNA.…”

“…As described previously, use of a lower salt concentration, that is, 10 mM Tris‐Cl (pH 8.5), resulted in minimal binding of pDNA to doxo‐beads (not shown), which further indicated minimal contribution of Coulombic interactions to the binding. On the other hand, berenil interacts with DNA using a combination of Coulombic, hydrogen bonding, and hydrophobic interactions . At high concentrations of ammonium sulfate, Coulombic interactions are largely shielded and the hydrophobic interactions may not be as strong as those with doxorubicin.…”

“…However, only 28% of pDNA was recovered from berenil‐beads under the same conditions (Figure C). It is likely that Coulombic interactions between pDNA and berenil‐beads at lower salt concentrations are responsible for the modestly higher retention of pDNA on berenil‐beads …”

“…In this study, doxorubicin, a commonly used anticancer drug was conjugated to aminoglycoside‐derived microbeads (Amikabeads) and the resulting materials were investigated for their ability to bind pDNA . The pDNA binding efficacy of doxorubicin‐conjugated microbeads was compared to that of microbeads derived with berenil, which is a known DNA‐binding pseudo‐affinity ligands . The efficacy of binding methylated as well as unmethylated pDNA was also investigated by processing plasmids through E. coli that are well known to either engender methylation of plasmids or not.…”

Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography

“…Chromatography is the most suitable method for this purpose [10,12], however, the poor selectivity that most of the matrices display towards pDNA its one of its major drawbacks [13]. The use of affinity ligands can be a simple and efficient approach to overcome this problem [14,15]. Affinity and pseudo-affinity chromatography use the specificity and bio-recognition properties of the ligands, based on specific and reversible interactions found in biological systems, to separate the pDNA molecules from the impurities found in cell lysates [16].…”