In Vitro Transcription of Modified mRNA

In vitro transcription was performed as previously described^{29 (link),30 (link)}. Briefly, plasmids except for that containing B18R gene was linearized with Spe-I, purified and used as template for in vitro transcription using T7 High Yield RNA Synthesis Kit (Cat #E2040, NEB) in the presence of anti-reverse cap analogue (Trilink) according to manufacturer’s protocol with a capping efficiency of ~80% (based on 4:1 ratio of ARCA cap to GTP). Pseudouridine (Ψ)-modified mRNA was synthesized by the same method with uridine triphosphate completely replaced by pseudouridine triphosphate (Trilink). Plasmid containing B18R gene was linearized with Sac-I, purified and transcribed. The transcript was then subjective to enzymatic poly A tail reaction with E.coli poly (A) polymerase (Cat #M0276, NEB). Successful poly A tailling was confirmed with gel electrophoresis. All synthesized mRNA was purified with RNeasy kit (Qiagen), quantified by spectrophotometry and analyzed by agarose gel electrophoresis to confirm the synthesis of full-length mRNA.