東京科学大学未来社会創成研究院地球生命研究所・特任准教授寺坂 尚紘Naohiro TerasakaEarth-Life Science Institute, Institute of Future Science, Institute of Science TokyoAbstractTherapeutic long RNAs, exemplified by mRNA vaccines, are difficult to be synthesized chemically and are thus produced via in vitro transcription using RNA polymerase. However, conventional in vitro transcription reactions suffer from the generation of byproducts such as double-stranded RNA and short RNA fragments. To introduce essential chemical structures for RNA pharmaceuticals, such as the 5’-cap, co-transcriptional capping strategies using nucleotide analogs have been developed. While this method is simpler than enzymatic post-transcriptional capping, it has drawbacks, including the low incorporation efficiency of nucleotide analogs, the requirement for large amounts of expensive analogs, and the unintended synthesis of uncapped RNA due to competition with natural NTPs. Uncapped RNA retains a 5’-triphosphate group, which can trigger immune responses. As a result, multiple steps of synthesis and purification are required to obtain high-purity capped mRNA, increasing production costs and causing batch-to-batch variability.はじめにmRNA ワクチンに代表される長鎖 RNA 医薬品は化学合成することが難しいため，RNA ポリメラ― 200 ―In this study, we aimed to develop RNA polymerase variants with enhanced transcription activity and improved incorporation of nucleotide analogs for application in RNA pharmaceutical production. To achieve high-throughput engineering of RNA polymerase, we established a molecular evolution platform combining microfluidic droplets and a cell-free translation system. A mutant library of RNA polymerase was constructed by random mutagenesis, and variants with elevated transcription activity were selected through directed evolution. As a result, we successfully obtained a mutant with higher transcription activity compared to the wild-type enzyme.高純度 mRNA 薬剤の合成を可能にする RNA ポリメラーゼの開発Engineered RNA polymerasesto produce therapeutic RNA molecules
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