Streamlined DNA template preparation and co-transcriptional 5′ capped RNA synthesis enabled by solid-phase catalysis

The success of SARS-CoV-2 mRNA vaccines demonstrated that rapid, large-scale manufacturing of synthetic mRNA is necessary for an effective and timely response to a pandemic. Innovations in areas such as template design and manufacturing processes are being implemented to facilitate more simple, cost-effective and scalable mRNA synthesis. In this study, for the first time, we demonstrate that the enzymatic steps in mRNA production (including DNA template linearization, RNA synthesis, 5′ capping and methylation) can be carried out using enzymes immobilized to a solid support. Specifically, we demonstrate efficient IVT template DNA linearization using immobilized BspQI, where the linearized template DNA can be directly used in IVT without the need of purification. We also showed that immobilized T7 RNA polymerase, Faustovirus RNA capping enzyme (FCE), vaccinia cap 2′-O-methyltransfease (2′OMTase) and a novel FCE::T7RNAP fusion enable efficient enzymatic synthesis of Cap-1 RNA in a one-pot format. This solid-phase enzymatic platform may enable highly efficient, seamless and continuous mRNA synthesis workflows that minimizes sample loss and units of operation in biopharmaceutical manufacturing. ### Competing Interest Statement G.G.M., M.S. and are current employees of New England Biolabs that markets molecular biology reagents. The affiliation does not affect the authors′ impartiality, adherence to journal standards and policies, or availability of data.