FADS and semi-rational design modified T7 RNA polymerase reduced dsRNA production, with lower terminal transferase and RDRP activities

T7 RNA polymerase (T7 RNAP) is the preferred tool for in vitro transcription (IVT), it synthesizes mRNA while accompanied by the generation of dsRNA by-products. This undesirable dsRNA triggers immune stress responses, compromises therapeutic efficacy, and raises safety concerns. To evolve T7 RNAP for reduced dsRNA, we pursued two complementary strategies. Firstly, the FADS (fluorescence-activated droplet sorting) based on molecular beacons was used to screen random libraries with diversity exceeding 105. Secondly, we constructed several single-site saturated libraries to facilitate the transition of T7 RNAP from the initiation to the elongation conformation. These libraries were screened using the traditional microplate-based dual-probe screening technique. Both approaches identified two dominant variants: Mut1 (V214A) and Mut7 (F162S/A247T) from FADS, Mut11 (K180E) and Mut14 (A70Q) from saturated libraries. Furthermore, the combinatorial mutant Mut17 (A70Q/F162S/K180E), generated via DNA shuffling, exhibited significantly reduced dsRNA production compared to the wild-type under various conditions, ranging from 0.18% to 1.80%, with a minimum value of 0.5 pg/g. Cell experiments confirmed that variants generated capped-mRNA with similar quality and quantity to the wild-type, while significantly reducing immune stress response in cells. These results indicate the compatibility and broad potential applications of these mutations. We then observed a close correlation between the production of dsRNA and the activities of T7 RNAP in terminal transferase and RDRP. Particularly, the terminal transferase activity appears to play a critical role in dsRNA generation. These findings align with the mechanism of dsRNA formation during IVT and provide new screening criteria for further evolution of T7 RNAP.