Engineered T7 RNA polymerase reduces dsRNA formation by lowering terminal transferase and RNA-dependent RNA polymerase activities.

T7 RNA polymerase (RNAP), the preferred tool for in vitro transcription (IVT), can generate double-stranded RNA (dsRNA) by-products that elicit immune stress and pose safety concerns. By combining the molecular beacon-based fluorescence-activated droplet sorting (FADS) utilized for random library screening with site-directed mutagenesis aimed at facilitating conformational changes in T7 RNAP, we successfully identified four mutants that exhibit reduced dsRNA content: M1 (V214A), M7 (F162S/A247T), M11 (K180E) and M14 (A70Q). Furthermore, the combinatorial mutant M17 (A70Q/F162S/K180E) exhibited significantly reduced dsRNA production under various conditions. Cellular experiments confirm the application potential of the mutants, displaying mitigated immune stress responses and enhanced protein translation compared to the wild-type protein. We then observed a close correlation between the production of dsRNA and the terminal transferase and RNA-dependent RNAP (RDRP) activities of T7 RNAP. The terminal transferase activity adds several nucleotides to the terminus of RNAs, while the RDRP activity extends the complementary region formed by self-pairing. In summary, we developed a novel approach for engineering T7 RNAP and demonstrated its potential in screening for T7 RNAP variants with reduced dsRNA production or improved product integrity.