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The recent application of histidine-agarose affinity supports in plasmid purification takes advantage of the biorecognition of nucleic acid bases by the histidine ligand. This consideration prompted the need for better understanding the interactions involved in affinity chromatography of plasmid DNA with the histidine-agarose support. In this work, we used synthetic homo-deoxyoligonucleotides with different sizes (1-30 nucleotides long), to explore the effect of several conditions like hydrophobic character of the individual bases, presence of secondary structures, temperature, pH and salt concentration on the mechanism of retention of nucleic acids to histidine-agarose support. One of the most striking results shows that histidine interacts preferentially with guanine, and the presence of secondary structures on polyA and polyG oligonucleotides has a significant influence on retention. Otherwise, the temperature manipulation has not shown a direct influence on oligonucleotide retention, only inducing conformational changes on secondary structures. Overall, the results obtained provide valuable information for the future development and implementation of histidine and other amino acids as ligands in chromatography for the purification of plasmid DNA and other nucleic acids, by improving the knowledge of the interactions involved as well as of the parameters influencing the retention.

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Chromatographic technology is undoubtedly one of the most diverse and powerful purification methods for downstream process applications. The diversity and quantity of biomolecules present in crude extracts as well as the similarities between impurities and the target biomolecule are considered the critical challenges in the extraction and purification steps. Thus, it is important to optimize the purification protocol to achieve maximum recovery of the target sample. The structure of chromatographic supports has been continuously developed to afford rapid and efficient separations, as well as, the application of specific ligands to improve the selectivity for the target molecule. The present review discusses the structural progress and evolution of the chromatographic supports that have been used for plasmid DNA purification. Nowadays, the most desirable form of plasmid for gene therapy and DNA vaccination is the supercoiled isoform, due to its stability and higher transfection efficiency over other plasmid topologies. However, the main challenge is not only to produce high quantities of supercoiled plasmid DNA but also to preserve its quality, meeting the strict requirements recommended by the regulatory agencies. Therefore, this review will focus on the chemical and structural classification of the different media and on some of the specific ligands used for plasmid DNA bioseparation.

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The development of a strategy to plasmid DNA (pDNA) purification has become necessary for the development of gene therapy and DNA vaccine production processes in recent years, since this nucleic acid and most of contaminants, such as RNA, genomic DNA and endotoxins, are negatively charged. An ideal separation methodology may be achieved with the use of affinity interactions between immobilized amino acids and nucleic acids. In this study, the binding behaviour of nucleic acids under the influence of different environmental conditions, such as the composition and ionic strength of elution buffer, and the temperature, is compared with various amino acids immobilized on chromatography resins. Supercoiled (sc) plasmid isoform was isolated with all matrices used, but in some cases preferential interactions with other nucleic acids were found. Particularly, lysine chromatography showed to be an ideal technology mainly on RNA purification using low salt concentration. On the other hand, arginine ligands have shown a greater ability to retain the sc isoform comparatively to the other nucleic acids retention, becoming this support more adequate to sc pDNA purification. The temperature variation, competitive elution and oligonucleotides affinity studies also allowed to recognize the dominant interactions inherent to biorecognition of pDNA molecule and the affinity matrices.