Multimodal chromatography of supercoiled minicircles: A closer look into DNA-ligand interactions

Abstract

This work describes a process for the purification of minicircles (MC), obtained from Escherichia coli by intramolecular recombination of a parental plasmid (PP) into a target MC and a miniplasmid (MP) molecule. The downstream process includes alkaline lysis, pre-purification by tandem precipitation and an endonuclease digestion step that specifically converts supercoiled (sc) MP into open circular (oc) MP. Next, a MM Capto adhere matrix is used with a NaCl gradient to isolate sc MC. Results show that oc species elute first (≈69 mS/cm), followed by sc (≈75 mS/cm) and RNA (≈140 mS/cm). Gel electrophoresis reveal that sc MC fractions are homogeneous (>90%) and impurity-free. A study of the underlying DNA-ligand interactions is also presented. Control experiments with reference matrices and the same gradient indicate that the anion-exchange (charged N) and hydrophobic moieties (phenyl) alone are unable to separate the nucleic acids. Nuclease sensitivity assays and bioinformatics analysis further revealed that base exposure is more likely in sc isoforms as a result of duplex destabilization and B-Z transitions. Based on the data, we suggest that binding of the charged nitrogen of the matrix to the phosphate backbone produces an asymmetry in charge neutralization that induces bending and base exposure. The exposed bases thus become available for cation-π, π-π stacking and H-bonding interactions, that reinforce the electrostatic binding and effectively produce a network of non-covalent bonds that effectively bind molecules to the matrix. The order of elution observed thus essentially reflects the increasing degree of base exposure in oc DNA, sc DNA and RNA. The NaCl-triggered elution of the different species results from the disruption of ionic interactions and concomitant decrease in base exposure and weakening of the bond network.