Publicação
Specific berenil–DNA interactions: an approach for separation of plasmid isoforms by pseudo–affinity chromatography
| Resumo: | Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the present study, pDNA binding affinities of berberine, berenil, kanamycin and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions varying the type and the salt concentration, and was performed both in absence and in presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand, showed a total retention of pDNA using 1.3 M ammonium sulphate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing salt concentration to 0.6 M and then to 0 M. These results suggest a promising application of berenil as ligand for specific pDNA supercoiled (sc) isoform purification by pseudo-affinity chromatography. |
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| Autores principais: | Caramelo-Nunes, C. |
| Outros Autores: | Tente, T.; Almeida, P.; Marcos, João Carlos; Tomaz, C. T. |
| Assunto: | Small DNA ligands Berenil Minor groove binder Intercalator Pseudo-affinity chromatography |
| Ano: | 2011 |
| País: | Portugal |
| Tipo de documento: | artigo |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the present study, pDNA binding affinities of berberine, berenil, kanamycin and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions varying the type and the salt concentration, and was performed both in absence and in presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand, showed a total retention of pDNA using 1.3 M ammonium sulphate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing salt concentration to 0.6 M and then to 0 M. These results suggest a promising application of berenil as ligand for specific pDNA supercoiled (sc) isoform purification by pseudo-affinity chromatography. |
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