Breaking barriers in the sensitive and accurate mass determination of large DNA plasmids by mass photometry

DNA plasmids (pDNA) 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. Furthermore, pDNA is used in CRISPR-based gene editing, RNA therapeutics, and DNA vaccines. Due to the rapidly increasing use and application of a wide variety of pDNA, analytical methods to characterize their key attributes are vital. Because of their high molecular weight, accurate and fast mass analyses of pDNA, as a measure of quality control, is rather challenging. 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%. Even though the landing of pDNA during MP analysis can be improved by using coated glass slides, the large dsDNA particles diffract light beyond the diffraction limit, rendering most landing events unusable. To overcome these issues, we introduce a fast (30 s) and simple protocol to convert dsDNA particles rapidly into ssDNA-like particles just prior to analysis and show that these particles behave nearly perfect for MP. Using this protocol accurate and correct masses of pDNAs can be obtained by MP, 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.