Building on its success against COVID-19, mRNA technology holds promise for novel vaccines and treatments targeting various diseases. However, a critical challenge remains: current in vitro transcription (IVT) methods used for mRNA synthesis produce harmful double-stranded RNA (dsRNA) byproducts, compromising therapeutic safety. While high-temperature IVT reduces these byproducts, it damages the activity of T7 RNAP.
Researchers predicted the thermally stable mutations in T7 RNAP through the PROSS server and adopted the greedy accumulation-directed evolution approach to optimize the multi-site combinatorial mutant, breaking through the melting temperature limit of T7 RNAP, enhancing its thermal stability, and reducing the level of dsRNA by-products.
Homology modeling and molecular dynamics simulations revealed the T7 RNAP mutant underwent overall conformational changes, alterations in helical propensity, and the formation of new salt bridges, providing efficient mRNA biocatalysts and offering a method for further T7 RNAP engineering.
The work entitled “Enhanced high-temperature performance of T7 RNA polymerase by greedy accumulation-directed evolution of thermostability” was published on Systems Microbiology and Biomanufacturing (published on May 19, 2025).
DOI: 10.1007/s43393-025-00370-3