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The messenger RNA mRNA 5 -cap structure is indispensable for mRNA translation initiation and stability Despite its importance large-scale production of capped mRNA through in vitro transcription IVT synthesis using vaccinia capping enzyme VCE is challenging due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation Here in the present study we found that the VCE together with 2 -O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition We have therefore designed an integrated purification and solid-based capping protocol which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3 -end poly-A in mRNA in situ capping of mRNA 5 -end by supplying the enzymes and subsequent eluting of the capped mRNA from the poly dT media Using mRNA encoding the enhanced green fluorescent protein as a model system we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency as compared with the conventional process which involved at least mRNA preseparation from IVT solution-based capping and post-separation and recovering steps Specifically the new process accomplished a 1 76-fold 84 21 over 47 79 increase in mRNA overall recovery a twofold decrease in operation time 70 vs 140 min and similar high capping efficiency both close to 100 Furthermore the solid-based capping process greatly improved mRNA stability such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase in contrast mRNA in the solution-based capping process degraded almost completely Meanwhile we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency enhanced mRNA recovery and improved stability against RNase therefore can act as a simple efficient and cost-effective platform technology suitable for large-scale production of capped mRNA 2023 Wiley Periodicals LLC

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On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and impro...

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with poly dT media as keyword