Abstract: The stability of supercoiled plasmid DNA (sc DNA) is a core issue in the development of nucleic acid vaccines, directly determining the efficacy and safety of the vaccines. This study investigated the effects of ultrafiltration membrane packs with different molecular weight cut-offs on the concentration and purification of plasmid DNA, and the effects of different stabilizers on the stability of plasmid DNA during purification process and storage were evaluated. The results showed that as the molecular weight cut-off of the ultrafiltration membrane pack increased, the yield of sc DNA significantly decreased, the damage to the supercoiled structure was exacerbated, the melting temperature (Tm) dropped, and the degradation rate during storage accelerated. These findings suggested that larger pore sizes in the membrane packs may lead to increased physical or chemical damage to sc DNA, such as double-strand breaks or the formation of open circular structures. Adding trisodium citrate during ultrafiltration significantly enhanced the stability of sc DNA, reducing losses during the ultrafiltration purification and subsequent storage processes. Molecular dynamics simulations further revealed that trisodium citrate formed a protective layer around DNA molecules through hydrogen bonds, effectively reducing damage to sc DNA during purification and storage. Additionally, the addition of trisodium citrate significantly increased the yield of mRNA after in vitro transcription but had no significant effect on the content of by-products such as dsRNA. Therefore, using a membrane pack with a smaller molecular weight cut-off (e.g., 30 kDa) in combination with trisodium citrate as a stabilizer can significantly improve the purification efficiency and stability of sc DNA. This finding provides theoretical support for optimizing the purification and storage conditions of plasmid DNA and lays an important foundation for the development of more stable and efficient DNA and mRNA vaccines, which is crucial for advancing nucleic acid-based therapeutic strategies.

Key words: supercoiled plasmid DNA, ultrafiltration, stability, trisodium citrate, molecular simulation

摘要： 超螺旋质粒DNA (sc DNA)的稳定性是核酸疫苗研发中的核心问题，直接决定了疫苗的功效性和安全性。本研究探讨了不同截留分子量的超滤膜包对质粒DNA浓缩纯化的影响，并评估了不同稳定剂在质粒DNA纯化及储存过程中对其稳定性的作用机制。结果表明，随着超滤膜包截留分子量增大，sc DNA的收率显著降低，超螺旋结构损伤加剧；通过差示扫描量热法(DSC)检测发现，其解链温度(Tm)下降，且在储存过程中降解速度加快。在超滤过程中添加柠檬酸三钠可显著增强sc DNA的稳定性，有效减少其在超滤纯化和后续储存过程中的损失。分子动力学模拟进一步揭示，柠檬酸三钠通过氢键在DNA分子周围形成保护层，从而有效缓解sc DNA在纯化和后续储存过程中的结构损伤。此外，添加柠檬酸三钠还可显著提高体外转录后mRNA的产量，但对副产物双链RNA (dsRNA)的含量无明显影响。因此，采用较小截留分子量的膜包(如30 kDa)并结合柠檬酸三钠作为稳定剂，可显著提升sc DNA的纯化效率和稳定性。本研究结果为优化质粒DNA的纯化和储存条件提供了理论支持，也为开发更稳定、高效的DNA和mRNA疫苗奠定了重要基础。

关键词: 超螺旋质粒DNA, 超滤, 稳定性, 柠檬酸三钠, 分子模拟