N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells

Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N(1-)Methylpseudouridine (m1 Psi) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stern from the high protein expression from ml containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1 Psi significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1 Psi modification have enormous potentials in the research and application of biofunctional RNA tools.