Analysis of encoded plasmid poly(dA) length distribution using liquid chromatography-mass spectrometry

Poly(A) tail plays critical roles in mRNA stability and translation efficiency, enhancing the potential of mRNA in vaccine and therapeutics. Encoded poly(dA) tail provides advantages over enzymatically synthesized tails, including product homogeneity, higher reproducibility, and reduced manufacturing costs. However, instability of homopolymeric regions during E. coli replication often leads to truncations, making accurate measurement of poly(dA) length essential for process optimization. Traditional methods such as Sanger sequencing are limited by labor-intensive workflows and low resolution of heterogeneous populations. Here, we developed a liquid chromatography-mass spectrometry (LC-MS) workflow to characterize encoded poly(dA) tail length distributions in plasmid DNA. In this study, two sample collection methods, polymerase chain reaction (PCR) and restriction enzyme digestion, were evaluated for LC-MS analysis. Both strategies achieved single-adenosine resolution and confirmed theoretical poly(dA) lengths. The PCR method required lower sample input but introduced shorter poly(dA) populations due to polymerase slippage, resulting in broader distributions. In contrast, the digestion method provided higher accuracy through an amplification-free process but required higher sample input and was limited by enzyme recognition sites. Agarose gel electrophoresis was applied as a preliminary assessment tool to estimate heterogeneity, while LC-MS enabled precise distribution profiling. The polydispersity index (PDI) was introduced to quantify tail heterogeneity, and the PDI values obtained from the two sample collection methods for LC-MS analysis showed the same trend as Sanger sequencing for stable and unstable poly (dA) tails. Overall, LC-MS combined with enzymatic digestion provides an accurate approach for investigating encoded poly(dA) length distribution, supporting mRNA drug development and quality control.