Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
J Sep Sci. 2010 Sep;33(17-18):2610-8. doi: 10.1002/jssc.201000347.
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.
近年来，随着基因治疗和 DNA 疫苗生产工艺的发展，开发一种质粒 DNA（pDNA）纯化策略变得非常必要，因为这种核酸和大多数污染物（如 RNA、基因组 DNA 和内毒素）都是带负电荷的。理想的分离方法可以利用固定化氨基酸和核酸之间的亲和相互作用来实现。在这项研究中，比较了在不同环境条件（如洗脱缓冲液的组成和离子强度以及温度）下核酸的结合行为与固定在色谱树脂上的各种氨基酸。所有使用的基质都可以分离超螺旋（sc）质粒异构体，但在某些情况下，发现与其他核酸存在优先相互作用。特别是，赖氨酸色谱显示出使用低盐浓度纯化 RNA 的理想技术。另一方面，精氨酸配体显示出比其他核酸保留更大的保留 sc 异构体的能力，使这种载体更适合 sc pDNA 的纯化。温度变化、竞争洗脱和寡核苷酸亲和研究也使我们能够识别与 pDNA 分子和亲和基质的生物识别相关的固有主导相互作用。
