EPO and anemia: Next, we tested whether hRNase 4 could be utilized to characterize and discriminate between IVT mRNAs fully substituted with m1Ψ or mo5U. To this end, we chose an 800 nt mRNA encoding Erythropoietin (EPO) as a model substrate, which has been previously employed for proof-of-concept mRNA-based interventions to treat anemia (33,34), as well as for mRNA LC–MS/MS sequence validation (13).