Ebberink Eduard H T M, Deslignière Evolène, Ruisinger Alisa, Nuebel Markus, Thomann Marco, Heck Albert J R
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, the Netherlands.
Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, the Netherlands.
Mol Ther Nucleic Acids. 2025 Jul 17;36(3):102632. doi: 10.1016/j.omtn.2025.102632. eCollection 2025 Sep 9.
DNA plasmids (pDNAs) are essential for gene cloning and protein expression, whereby engineered plasmids serve as vectors to insert foreign DNA into host cells, enabling mass production of proteins and vaccines. Due to the rapidly increasing use and application of a wide variety of pDNA (e.g., CRISPR-based gene editing, RNA therapeutics, and DNA vaccines), analytical methods to characterize their key attributes are vital. Here we explore mass photometry (MP) to analyze pDNAs and find that it completely fails using standard procedures as developed for MP on proteins, with masses underestimated by 30%-40%. While DNA can be measured by using coated glass slides, the large double-stranded DNA (dsDNA) particles diffract light beyond the diffraction limit, rendering most landing events unusable. To overcome such issues, we introduce a formic acid-based denaturation protocol to convert dsDNA particles rapidly (∼30 s) into single-stranded DNA (ssDNA)-like particles and show that these particles behave nearly perfect for MP. Using this protocol accurate and correct pDNA masses can be obtained, with values within 1-3% of the expected mass. Using this protocol, MP can be used to mass analyze pDNA constructs from 1 to 15 MDa, suggesting that this approach may be widely adopted within academia and biopharma for essentially all plasmids.
DNA质粒（pDNA）对于基因克隆和蛋白质表达至关重要，通过工程化质粒作为载体将外源DNA插入宿主细胞，从而实现蛋白质和疫苗的大规模生产。由于各种各样的pDNA（例如基于CRISPR的基因编辑、RNA疗法和DNA疫苗）的使用和应用迅速增加，表征其关键属性的分析方法至关重要。在这里，我们探索了质量光度法（MP）来分析pDNA，发现按照用于蛋白质MP的标准程序完全无法成功，质量被低估了30%-40%。虽然可以使用涂覆的载玻片测量DNA，但大的双链DNA（dsDNA）颗粒使光的衍射超出衍射极限，导致大多数着陆事件无法使用。为了克服这些问题，我们引入了一种基于甲酸的变性方案，将dsDNA颗粒快速（约30秒）转化为类似单链DNA（ssDNA）的颗粒，并表明这些颗粒在MP中表现近乎完美。使用该方案可以获得准确且正确的pDNA质量，其值在预期质量的1%-3%范围内。使用该方案，MP可用于对1至15 MDa的pDNA构建体进行质量分析，这表明该方法可能在学术界和生物制药领域被广泛采用，用于基本上所有的质粒。
