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- Arginine homopeptides for plasmid DNA purification using monolithic supportsPublication . Cardoso, Sara; Sousa, Ângela; Queiroz, João; Azzoni, Adriano; Sousa, FaniPurification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.
- Arginine and di-arginine ligands for plasmid DNA purification using negative chromatographyPublication . Cardoso, Sara; Filho, Pedro De Alcântara Pessôa; Sousa, Fani; Azzoni, AdrianoThe increasing number of applications requiring highly purified plasmid DNA (pDNA) generates a corresponding need for simple, scalable, and cost-effective purification processes. Due to the pDNA large size and complex shape, the use of commercial chromatographic beads often results in poor yields and low binding capacities when operated in a positive mode. An alternative to overcome this limitation is the design of chromatographic ligand-resin systems able to efficiently operate in negative mode, where host impurities (especially low molecular weight RNA) are efficiently captured and separated from the target pDNA. In this work, arginine amino acid and di-arginine peptide (arginine-arginine) were immobilized in agarose resins and evaluated for negative chromatographic purification of pDNA from bacterial cell lysates. The results showed that RNA was preferentially bound to the ligands, interfering with the binding of pDNA. The amount of plasmid processed per column volume by arginine and di-arginine, under negative mode, was substantially larger comparing with the conventional positive mode, resulting in pDNA recoveries up to 99%, with a considerable reduction of host impurities. This study shows that negative mode chromatography using arginine-based ligands poses as an interesting alternative for intermediate and polishing pDNA purification operations, with considerable economic and environmental advantages.