Chemoaffinity Material for Plasmid DNA Analysis by High-Performance Liquid Chromatography with Condition-Dependent Switching between Isoform and Topoisomer Selectivity

Abstract

Plasmid DNA may exist in three isoforms, the linear, open-circular (oc, "nicked"), and covalently closed circular (ccc, "supercoiled") form. We have recently reported on the chromatographic separation of supercoiled plasmid topoisomers on cinchona-alkaloid modified silica-based stationary phases. Herein, we present a selectivity switching mechanism to achieve separation of isoforms and/or supercoiled topoisomers using the very same chromatographic column and system. While salt gradient elution facilitates topoisomer separation, the supercoiled species are eluting as a single peak upon elution by a mixed pH and organic modifier gradient, still well separated from the other isoforms. We have found that a mobile phase pH value near the pI of the zwitterionic adsorbent surface leads to full recovery of all plasmid DNA isoforms, which is a major issue when using anion exchange-based resins. Furthermore, the observed elution pattern, oc < linear < ccc, is constant upon changes of mobile phase composition, gradient slope, and plasmid size. The remarkable isoform selectivity found on quinine-based selectors is explained by van't Hoff plots, revealing a different binding mechanism between the supercoiled plasmid on one hand and the oc and linear isoforms on the other hand.

Comparative DNA isolation behaviours of silica and polymer based sorbents in batch fashion: monodisperse silica microspheres with bimodal pore size distribution as a new sorbent for DNA isolation

“…Polyacrylate/methacrylate-based sorbents have been widely used for DNA isolation due to their high specific surface area, suitable hydrophilicity and functional groups that can create effective reversible interaction with DNA [14][15][16][17][18][19]. Methacrylate based monolith was prepared by bearing diethylaminoethyl (DEAE) and butyl groups and used as a sorbent for selective extraction of DNA [20].…”

Substrate-source flexibility of an exponential-fed perfusion process to produce plasmid DNA for use as leishmaniasis vaccine

“…First-generation vaccines are typically produced using eukaryotic cells, while pDNA is manufactured using a bacterial host in high cell density cultures (HCDC), resulting in improved production systems [18]. Plasmid solutions obtained in Escherichia coli cultures contain supercoiled (CCC), open circular (OC) and linear (L) isoforms [19]. Isoform CCC pDNA is recommended by regulatory agencies for vaccine treatments due to the structural alterations present in the OC and L isoforms [20].…”

A rapid high-resolution method for resolving DNA topoisomers

“…In an alternative version, an oligonucleotide is labelled with a fluorescent dye and the readout is fluorescence anisotropy [ 13 ]; in this case, triplex formation occurs preferentially with relaxed DNA. A novel method for resolving topoisomers of DNA has been described involving quinine carbamate ligands attached to a silica gel [ 14 ]. This matrix can be utilised to achieve rapid and efficient preparation of DNA topoisomers.…”