E of anion-exchange chromatography of DNA using compaction agents

AbstractThe use of adsorptive chromatography for preparative nucleic acid separations is often limited by low capacity. Thepossibility that the adsorbent surface area sterically accessible to nucleic acid molecules could be increased by reducing theirradius of gyration with compaction agents has been investigated. The equilibrium adsorption capacity of Q Sepharoseanion-exchange matrix for plasmid DNA at 600 mM NaCl was enhanced by up to ca. 40% in the presence of 2.5 mMspermine. In addition, compaction agent selectivity has been demonstrated. Spermine, for example, enhances the adsorptionof both plasmid and genomic DNA, spermidine enhances binding only of plasmid, and hexamine cobalt enhances only thebinding of genomic DNA. Compaction may be generally useful for enhancing adsorptive separations of nucleic acids. 2002 Elsevier Science B.V. All rights reserved.Keywords:Adsorption; Compaction agents; Preparative chromatography; DNA; Spermine; Spermidine; Hexamine cobalt 1. Introduction on an anion-exchange column [5]. The main draw-back to this method is the low loading capacity ofThe increasing importance of nucleic acids in typical matrices, which typically adsorb ca. 0.5–1.0medicine and biological sciences [1–4] is driving mg plasmid DNA per ml in contrast to 10–100 mgdemand for improved methods of separation of DNA per ml for proteins.from RNA, proteins, and other contaminants. Anion- We speculated that the chromatographic separationexchange chromatography is widely used for purifi- of nucleic acids could be improved using compactioncation of RNA and DNA, with plasmid DNA agents. Compaction agents are involved in vivo inremaining adsorbed in high salt (usually 0.65–0.75 regulating cell growth and differentiation and at highM NaCl) while proteins and RNA flow through the concentration can reduce the volume occupied bycolumn. With a proper gradient, it is also possible to DNA by 10 –10 [6,7]. Well-characterized compac-