Synthesis of Modified RNA for Protein Expression

ModRNAs of the selected cardiogenic and vasculogenic growth factors were produced as previously described^{17 (link)}. Briefly, mRNA was synthesized using T7 RNA polymerase-mediated IVT from a linearized DNA template containing the target gene’s ORF, flanking 5′ and 3′ UTRs and a poly-A tail. A Cap1 structure was enzymatically added to the 5′ end to produce the final mRNA. Uridine was completely substituted with N1-methylpseudouridine to reduce potential immunostimulatory activity and to improve protein expression relative to unmodified mRNA. ModRNA was then purified using the Ambion MEGAclear kit and treated with Antarctic phosphatase (New England Biolabs) for 30 min at 37 °C to remove residual 5’-phosphates. All modRNAs were re-purified and quantified using Nanodrop (Thermo Scientific). After purification, modRNA was resuspended in MEGAclear buffer at 1 μg/μL concentrations and frozen at −80 °C for future use.