N¹-methyl-pseudouridine is incorporated with higher fidelity than pseudouridine in synthetic RNAs

In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality. To overcome the inherent immunogenicity, as well as to increase the therapeutic efficacy of the molecules, uridine analogs-such as pseudouridine (Ψ) and N¹-methyl-pseudouridine (m1Ψ), are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, we compared the incorporation fidelity of uridine analogs with different RNA polymerases. We demonstrate that m1Ψ is incorporated with higher fidelity than Ψ. The fidelity of nucleotide incorporation differs between RNA polymerases; however, the spectrum of mutations observed between the RNAPs is similar. We also show that the array of nucleotide misincorporation is not dependent on the template DNA sequence context and that the distribution of these misincorporated nucleotides is not localized to any specific region along the length of the RNA. Based on our findings, we introduce a novel method to improve uridine analog incorporation fidelity during IVT. Our proof-of-concept experiments for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing RNAPs for the generation of modified uridine-containing mRNAs in vitro.