Improving the fidelity of uridine analog incorporation during in vitro transcription.

In vitro transcribed synthetic messenger RNAs (mRNAs) represent a novel therapeutic modality and are currently being evaluated for a wide range of clinical indications. To overcome the inherent immunogenicity of the synthetic mRNAs, as well as to increase the therapeutic efficacy of the molecules, RNA sequence optimization is routinely performed and modified uridine analogs - such as pseudouridine and N1-methyl-pseudouridine, are incorporated in the synthetic mRNA. To decipher the fidelity with which these modifications are incorporated during the in vitro transcription (IVT) process, here, we compared, the incorporation fidelity of uridine, pseudouridine, or N1-methyl-pseudouridine in multiple RNA sequences with different single-subunit DNA-dependent RNA polymerases (ssRNAPs). By comparing the incorporation of each modified base to that of the unmodified equivalent, we demonstrate that N1-methyl-pseudouridine is incorporated with higher fidelity than pseudouridine. Furthermore, the various ssRNAPs exhibit different error rates; 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 protocol to improve uridine analog incorporation - without affecting total RNA yield - during IVT. Our proof-of-concept experiments and protocol for higher-fidelity incorporation of uridine analogs during IVT provide guidelines when choosing ssRNAPs for the generation of modified uridine-containing mRNAs in vitro. ### Competing Interest Statement The authors have declared no competing interest.