A review of advances in in vitro RNA preparation by ssRNAP

In vitro transcription (IVT) based on single-subunit RNA polymerase (ssRNAP) has enhanced the widespread application of RNA drugs in the biomedical field, showcasing unprecedented potential for disease prevention and treatment. While the classical enzyme T7 RNA polymerase (T7 RNAP) has driven significant progress in RNA production, several challenges persist. These challenges include the selectivity of the initiation nucleotide, low incorporation efficiency of modified nucleotides, limited processivity on certain templates, heterogeneity at the 3' end of RNA products, and high level of double-stranded RNA (dsRNA) byproducts. No review has systematically addressed the efforts to overcome these challenges. To fill this gap, we reviewed recent advances in engineering T7 RNAP variants and the discovery of novel ssRNAPs aimed at addressing the shortcomings of T7 RNAP. We also discussed the underlying mechanisms of ssRNAP-mediated byproduct formation, strategies to mitigate dsRNA production using modified nucleotides, and for the first time to sorted out the application of artificial intelligence in IVT. Overall, this review summarizes the advances in RNA synthesis via IVT and provides potential strategies for improving RNA products. We believe that ssRNAPs with more excellent performance will be on the stage of RNA synthesis in the near future to meet the growing demands of both scientific research and pharmaceutical industry.