Bundesanstalt für Materialforschung und Prüfung, 12205, Berlin, Germany.
Freie Universität Berlin, Institut für Chemie, 14195, Berlin, Germany.
Chembiochem. 2022 Oct 19;23(20):e202200391. doi: 10.1002/cbic.202200391. Epub 2022 Sep 14.
DNA long-term stability and integrity is of importance for applications in DNA based bio-dosimetry, data-storage, pharmaceutical quality-control, donor insemination and DNA based functional nanomaterials. Standard protocols for these applications involve repeated freeze-thaw cycles of the DNA, which can cause detrimental damage to the nucleobases, as well as the sugar-phosphate backbone and therefore the whole molecule. Throughout the literature three hypotheses can be found about the underlying mechanisms occurring during freeze-thaw cycles. It is hypothesized that DNA single-strand breaks during freezing can be induced by mechanical stress leading to shearing of the DNA molecule, by acidic pH causing damage through depurination and beta elimination or by the presence of metal ions catalyzing oxidative damage via reactive oxygen species (ROS). Here we test these hypotheses under well defined conditions with plasmid DNA pUC19 in high-purity buffer (1xPBS) at physiological salt and pH 7.4 conditions, under pH 6 and in the presence of metal ions in combination with the radical scavengers DMSO and Ectoine. The results show for the 2686 bp long plasmid DNA, that neither mechanical stress, nor pH 6 lead to degradation during repeated freeze-thaw cycles. In contrast, the presence of metal ions (Fe ) leads to degradation of DNA via the production of radical species.
DNA 的长期稳定性和完整性对于基于 DNA 的生物剂量学、数据存储、药物质量控制、供精和基于 DNA 的功能纳米材料等应用非常重要。这些应用的标准方案涉及 DNA 的反复冻融循环，这可能会对碱基、糖磷酸骨架以及整个分子造成有害的损伤。在文献中，可以找到三种关于冻融循环过程中发生的潜在机制的假设。有人假设，冷冻过程中 DNA 的单链断裂可能是由机械应力引起的，导致 DNA 分子的剪切，也可能是由于酸性 pH 值通过脱嘌呤和β消除造成损伤，或者是由于金属离子通过活性氧物种 (ROS) 催化氧化损伤。在这里，我们在生理盐和 pH 值为 7.4 的条件下，在高纯度缓冲液（1xPBS）中用质粒 DNA pUC19 在定义明确的条件下测试这些假设，在 pH 值为 6 且存在金属离子的条件下，与自由基清除剂 DMSO 和 Ectoine 一起测试。结果表明，对于 2686 个碱基对的长质粒 DNA，反复冻融循环既不会导致机械应力，也不会导致 pH 值下降导致降解。相比之下，金属离子（Fe）的存在会通过产生自由基物质导致 DNA 降解。
