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 (psi) and N-1-methyl-pseudouridine (m1 psi), 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 m1 psi is incorporated with higher fidelity than psi. 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.

Automated summary

In vitro transcribed synthetic mRNAs are a new therapeutic modality. Uridine analogs are incorporated to enhance therapeutic efficacy and overcome immunogenicity. This study compared the fidelity of uridine analog incorporation using different RNA polymerases and found that m1 psi has higher fidelity than psi. The spectrum of mutations differs between RNA polymerases, but the distribution is not dependent on DNA sequence context.