核糖核酸
产量(工程)
核苷
乙酸钠
DNA
体外
化学
抄写(语言学)
镁
焦磷酸酶
生物
生物化学
酶
色谱法
基因
材料科学
有机化学
冶金
语言学
哲学
作者
Karnyart Samnuan,Anna K. Blakney,Paul F. McKay,Robin J. Shattock
标识
DOI:10.1101/2021.01.08.425833
摘要
Abstract Self-amplifying RNA (saRNA) vaccines are able to induce a higher antigen-specific immune response with a more cost-effective and rapid production process compared to plasmid DNA vaccines. saRNAs are synthesized through in vitro transcription (IVT) however; this process has mainly been optimized for relatively short mRNAs. Here, we optimized the IVT process for long saRNAs, approximately 9.4 kb through a design of experiment (DoE) approach to produce a maximal RNA yield and validated the optimal IVT method on various sizes of RNA. We found that magnesium has the highest impact on RNA yield with acetate ions enabling a higher yield than chloride ions. In addition, the interaction between magnesium and nucleoside triphosphates (NTPs) is highly essential for IVT. Further addition of sodium acetate (NaOAc) during IVT provided no added benefit in RNA yield. Moreover, pyrophosphatase was not essential for productive IVT. The optimal IVT method can be used to synthesize different lengths of RNA. These findings emphasize the ability to synthesize high quality and quantity of saRNA through IVT and that the optimal amount of each component is essential for their interactions to produce a high RNA yield.
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