I have been following the IVT protocol as described in Henderson et al., “Cap 1 Messenger RNA Synthesis with Co-Transcriptional CleanCap® Analog by In Vitro Transcription.” This involves a 2.5hr incubation at 37C during the IVT reaction. Preforming the IVT protocol using the standard nucleic acids produces good results and a high yield of mRNA (5ug/ul). However, when I try to substitute uridine for pseudouridine, following the same protocol, I get very little (~10-20ng/ul) mRNA.

Is creating U-modified synthetic mRNA as simple as subbing out uridine for pseudouridine, or am I missing something? Why could my pseudouridine IVT fail while my regular IVT works just fine?