Development of a WT RNAP-based kit for high-yield synthesis of chemically modified RNA

ABSTRACT With the growing number of RNA therapeutics approved for clinical uses and the fundamental advantages of using RNA in regulating protein expression, vaccines, genome editing, and targeting ligands, RNA has attracted substantial recent interest from both academic institutions and the pharmaceutical industry. Unless the RNA sequences of interest are very short (< 60 bp), in vitro transcription is often the desired approach for synthesis. Due to their inherent instability against enzyme degradation, RNAs for use in biotechnology and medicine require chemical modifications. Conventional T7 RNA polymerase (T7RNAP) is highly efficient in making long RNAs, but it has been difficult to incorporate chemically modified nucleotides. In contrast, T7RNAP mutants can incorporate chemically modified nucleotides but the yields of transcription are significantly lower, driving up the costs for both biomedical research and healthcare. By investigating the key parameters that influence T7RNAP activity, we have identified the barriers that hinder wild-type T7RNAP from accepting modified substrates and developed formulations and protocols to address them. Based on these innovations, here, we propose to develop a new in vitro transcription kit that combines the benefits of both worlds, the high efficiency and low cost of wild- type T7RNAP, and the tolerance of modified nucleotides of mutant T7RNAPs. This kit will generate chemical- modified serum-stable RNAs with a minimum 10-fold increase in yield over current best-performing kits, thus drastically improving the affordability of current and future RNA drugs.