Identification of Phage RNA Polymerases That Minimize Double-Stranded RNA By-Product Formation and Their Characterization via In Vitro Transcription

Therapeutics based on RNA are commonly produced via biocatalytic approaches using RNA polymerases. The most frequently applied enzyme is the RNA polymerase of Enterobacteria phage T7. However, this enzyme has unfavorable properties, like the formation of double-stranded RNA (dsRNA). This undesired by-product can activate the innate immune system via pattern recognition receptors and cause inflammation. Removal of the contaminant is time-consuming and expensive. In this work, we applied a genome mining approach to identify unidentified single-subunit RNA polymerases with minimal dsRNA generation. A large meta database was screened, and 74 sequences were selected. Two RNA polymerases generating barely detectable amounts of dsRNA were identified from the initial sequence portfolio. Their promoters were detected via a fluorescent RNA aptamer screening, and slightly acidic transcription conditions were established. Further activity characterization showed a significant reduction of dsRNA to 0.001% and 0.02%. Due to these beneficial attributes, these RNA polymerases generate mRNA with enhanced stability, which most likely lowers the immune response towards the desired mRNA. This could be especially useful for producing long RNAs, such as self-amplifying RNA, as these typically require improved stability and low dsRNA content.