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首页> 外文期刊>Journal of pharmaceutical sciences. >Evaluation of the effect of non-B DNA structures on plasmid integrity via accelerated stability studies.
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Evaluation of the effect of non-B DNA structures on plasmid integrity via accelerated stability studies.

机译：通过加速稳定性研究评估非B DNA结构对质粒完整性的影响。

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摘要

Plasmid biopharmaceuticals are a new class of medicines with an enormous potential. Attempts to increase the physical stability of highly purified supercoiled (SC) plasmid DNA in pharmaceutical aqueous solutions have relied on: (i) changing the DNA sequence, (ii) improving manufacturing to reduce deleterious impurities and initial DNA damage, and (iii) controlling the storage medium characteristics. In this work we analyzed the role of secondary structures on the degradation of plasmid molecules. Accelerated stability experiments were performed with SC, open circular (OC) and linear (L) isoforms of three plasmids which differed only in the "single-strandlike" content of their polyadenylation (poly A) signals. We have proved that the presence of more altered or interrupted (non-B) DNA secondary structures did not directly translate into an easier strand scission of the SC isoforms. Rather, those unusual structures imposed a lower degree of SC in the plasmids, leading to an increase in their resistance to thermal degradation. However, this behavior was reversed when the relaxed or L isoforms were tested, in which case the absence of SC rendered the plasmids essentially double-stranded. Overall, this work suggests that plasmid DNA sequence and secondary structures should be taken into account in future investigations of plasmid stability during prolonged storage.
机译：质粒生物药物是一类具有巨大潜力的新型药物。试图提高药物水溶液中高纯度超螺旋（SC）质粒DNA的物理稳定性的努力有赖于：（i）改变DNA序列，（ii）改进生产工艺以减少有害杂质和初始DNA损伤，以及（iii）控制存储介质的特性。在这项工作中，我们分析了二级结构在质粒分子降解中的作用。使用三个质粒的SC，开环（OC）和线性（L）亚型进行了加速的稳定性实验，它们的聚腺苷酸化（poly A）信号的“单链状”含量不同。我们已经证明存在更多改变或间断的（非B）DNA二级结构并不能直接转化为更容易的SC同工型链断裂。而是，那些不寻常的结构在质粒中施加了较低的SC，导致它们对热降解的抗性增加。然而，当测试松弛或L同工型时，这种行为被逆转，在这种情况下，SC的缺失使得质粒基本上是双链的。总的来说，这项工作表明在长期保存期间对质粒稳定性的进一步研究中应考虑质粒DNA序列和二级结构。

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