High-throughput monitoring of free Mg2+ in IVT reaction reveals effects of free Mg2+ on mRNA synthesis and DNA digestion.

mRNA vaccines and therapeutics are produced with an in vitro transcription reaction (IVT), a condensation of nucleoside triphosphates (NTPs) into a nascent mRNA chain templated by DNA and catalyzed by RNA polymerase. While tools exist to monitor NTP consumption, the role of the essential cofactor Mg2+ in IVT has not been extensively studied due to the lack of suitable analytical methods. In this study, we report the development of a high-throughput analytical method for monitoring free Mg2+ using selective fluorescent indicators. We combine this free Mg2+ monitoring method with previously reported rapid at-line monitoring of NTP and mRNA concentrations and apply it in batch and fed-batch IVT systems, varying Mg:NTP ratios and NTP concentrations to assess their effects on mRNA yield via free Mg2+. We experimentally demonstrate that free Mg2+ is critical for efficient mRNA production, with low free Mg2+ correlating with slow reaction progression or reaction stalling. For the first time, we experimentally show that under certain IVT conditions, the released free Mg2+ can be effectively utilized to continue the IVT reaction by feeding NTPs only. We also show that pyrophosphatase concentration significantly affects free Mg2+ levels and relate this to IVT kinetics and dsRNA content. Additionally, we demonstrate for the first time that free Mg2+ affects downstream DNase digestion of the pDNA template, resulting in residual pDNA when free Mg2+ levels are insufficient for DNase activity. Our findings provide an accessible methodological platform to improve the efficiency of mRNA production by optimizing IVT conditions and ensuring DNase activity.