Molecular therapy. Nucleic acidsAug 8, 2025

Accurately measuring large DNA plasmids using mass photometry

Updated Jan 2, 2026

Abstract

Mass photometry can accurately measure plasmid DNA (pDNA) masses within 1-3% of expected values using a new formic acid-based denaturation protocol.

Standard mass photometry techniques significantly underestimate pDNA masses by 30%-40%.
Large double-stranded DNA particles diffract light beyond the diffraction limit, which leads to unusable measurement events.
The new protocol converts double-stranded DNA into single-stranded DNA-like particles in approximately 30 seconds.
This method enables accurate mass analysis of pDNA constructs ranging from 1 to 15 MDa.
The approach may facilitate widespread adoption of mass photometry in academia and biopharma for plasmid analysis.

Simplified

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.

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