Enhanced high-temperature performance of T7 RNA polymerase by greedy accumulation-directed evolution of thermostability

Abstract

T7 RNA polymerase (T7 RNAP)-catalyzed in vitro transcription (IVT) is the gold standard manufacturing process for large-scale production of therapeutic mRNA molecules. However, the undesired catalytic activity of T7 RNAP concomitantly generates deleterious impurities, such as double-stranded RNAs, that can exacerbate the downstream purification burden and engender safety concerns. The aim of this study was to engineer T7 RNAP thermostability for high-temperature IVT to reduce the dsRNA. The web server PROSS was utilized to predict thermostable mutation sites from the intermediate and elongation structure of T7 RNAP. Through systematic evaluation of individual mutation sites followed by greedy-accumulation optimization of multi-site combinatorial mutants, we successfully overcame the inherent activity-stability trade-off during the evolution and obtained a thermostable variant, M10 (T_m: 49.5°C), which exhibits robust catalytic activity at elevated temperatures and significantly reduced dsRNA byproduct formation. Structural analysis using homology modelling and molecular dynamics (MD) simulation revealed that the accumulated mutations increased the local rigidity of T7 RNAP with a compacted conformation, enhanced the helical propensity, and allowed the formation of new salt bridges. The enhanced mutant has the potential to act as an effective biocatalyst for high-temperature IVT, adding in high-quality mRNA production, which is a prerequisite for optimizing the downstream purification processes and improving the clinical viability of such therapeutic agents.

The PROSS sever was used to predict thermostable mutations in T7 RNAP. A greedy-accumulation approach led to the creation of multi-site combinatorial mutants of T7 RNAP, exhibiting robust catalytic activity at higher temperatures and significantly lower levels of dsRNA byproducts

Keywords

T7 RNAP / In vitro transcription / Thermostability / mRNA therapeutics / dsRNA

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Funding

Social Development Key Project of Jiangsu Province(BE2023682)

RIGHTS & PERMISSIONS

Jiangnan University