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柱上聚 dT 介导的 mRNA 封端可实现高效率封端、提高 mRNA 回收率,并改善其对核糖核酸酶的稳定性。

On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

机构信息

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China.

Key Laboratory of Biopharmaceutical Preparation and Delivery, Chinese Academy of Sciences, Beijing, China.

出版信息

Biotechnol Bioeng. 2024 Jan;121(1):206-218. doi: 10.1002/bit.28560. Epub 2023 Sep 25.

Abstract

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.

摘要

信使 RNA(mRNA)5' 帽结构对于 mRNA 翻译起始和稳定性是必不可少的。尽管其重要性不言而喻,但由于需要繁琐的预分离和后分离步骤,导致 mRNA 损失和降解,因此通过痘苗病毒加帽酶(VCE)进行体外转录(IVT)合成来大规模生产加帽 mRNA 具有挑战性。在这里,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地催化聚 dT 介质连接的 mRNA 的加帽反应,生成具有帽 1 结构的 mRNA。因此,我们设计了一种集成的纯化和固载加帽方案,其中包括通过聚 dT 介质与 mRNA 的 3' 端多-A 的亲和结合来捕获 IVT 系统中的 mRNA,通过供应酶原位加帽 mRNA 5' 端,然后从聚 dT 介质中洗脱加帽的 mRNA。使用编码增强型绿色荧光蛋白的 mRNA 作为模型系统,我们证明与传统的方案相比,该新策略极大地简化了 mRNA 的制造过程并提高了整体回收率,而不牺牲加帽效率。传统方案至少涉及从 IVT 中分离 mRNA、基于溶液的加帽以及后续分离和回收步骤。具体而言,新工艺使 mRNA 整体回收率提高了 1.76 倍(84.21%比 47.79%),操作时间减少了两倍(70 分钟比 140 分钟),并且加帽效率相似(均接近 100%)。此外,固载加帽过程大大提高了 mRNA 的稳定性,即使在外源添加 RNase 的情况下,加帽过程中 mRNA 的完整性也能得到很好的保持;相比之下,溶液加帽过程中的 mRNA 几乎完全降解。同时,我们证明了该策略可以在批处理模式和柱上连续模式下运行。本工作的结果表明,这里开发的新型柱上加帽工艺可以实现高效率的加帽、提高 mRNA 的回收率以及提高对 RNase 的稳定性;因此,可以作为一种简单、高效、经济有效的平台技术,适用于大规模生产加帽 mRNA。

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