信使RNA(mRNA)5'-帽结构对于mRNA翻译的启动和稳定性是必不可少的。尽管它很重要,使用牛痘加帽酶(VCE)通过体外转录(IVT)合成大规模生产加帽mRNA是具有挑战性的,由于需要繁琐和多个前后分离步骤,导致mRNA丢失和降解。在目前的研究中,我们发现VCE与2'-O-甲基转移酶一起可以在优化条件下有效催化polydT介质束缚的mRNA的加帽,产生具有cap-1结构的mRNA。因此,我们设计了一个集成的纯化和基于固体的封端方案,涉及通过使用polydT培养基通过其与mRNA中的3'末端poly-A的亲和力结合从IVT系统中捕获mRNA,通过提供酶对mRNA5'端进行原位加帽,以及随后从聚dT培养基中洗脱加帽的mRNA。以编码增强型绿色荧光蛋白的mRNA为模型系统,我们已经证明,新策略大大简化了mRNA制造过程,并在不牺牲加帽效率的情况下提高了其整体回收率,与常规工艺相比,至少涉及IVT的mRNA预分离,基于解决方案的封盖,以及分离后和恢复步骤。具体来说,新的过程完成了1.76倍(84.21%比47.79%)mRNA的整体恢复增加,手术时间减少了两倍(70vs.140分钟),和类似的高封端效率(均接近100%)。此外,基于固体的加帽过程大大提高了mRNA的稳定性,这样即使在外源添加RNase的情况下,在加帽过程中也可以很好地保持mRNA的完整性;相反,基于溶液的加帽过程中的mRNA几乎完全降解。同时,我们表明,这种策略可以在间歇模式和柱上连续模式下操作。在这项工作中提出的结果表明,这里开发的新的柱上封端工艺可以实现高封端效率,增强mRNA恢复,并提高了抗RNase的稳定性;因此,可以作为一个简单的,高效,和适合大规模生产加帽mRNA的经济高效的平台技术。
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.