Deciphering the Mechanism: Overcoming Incompatibility for Continuous Cap-1 mRNA Synthesis via Multienzyme Cascades

Efficient synthesis of cap-1 mRNA is limited by low recovery and degradation risks in conventional multistep enzymatic reactions which require multiple intermediate separations. Here, we execute a mechanism-driven one-pot multienzyme cascade catalyzed by T7 RNA polymerase (T7-RNAP), vaccinia capping enzyme (VCE), and 2′-O-methyltransferase (2′-O-MTase) for continuous synthesis of cap-1 mRNA, where competitive interactions between Mg 2+ and VCE activity are deciphered. It is generally regarded that excess Mg 2+ promotes transcription but suppresses the capping; however, in our integrated cascade system, VCE exhibits largely enhanced tolerance to Mg 2+, which was uncovered due to the competitive binding effects of the transcription complex consisting of linearized plasmid DNA and T7-RNAP. By combining this mechanistic insight with programmed temperature regulation, we reconciled the conflicting requirements of IVT synthesis and capping of mRNA: temperature optimally balances T7-RNAP/VCE activity and 5′-end mRNA accessibility, while Mg 2+ plays a manifold role in the mRNA folding, Mg 2 PPi precipitation, transcription complex formation, and VCE-Mg-GTP activation. Importantly, our results demonstrate that supplementing Mg 2+ not only sustains the IVT reaction but also improves the capping efficiency without intermediate purification. These findings provide mechanistic insights into multienzyme incompatibility and its resolution, offering a promising application in continuous mRNA manufacturing with high efficiency at reduced process cost.