In vitro Synthesis of Capped and Modified mRNA

TRAIL- and PTEN-bearing mRNAs were generated by in vitro transcription. The human 5′UTR with Kozak sequence and 3′UTR sequence were synthesized commercially by Integrated DNA Technologies (Coralville, Iowa) and sub-cloned into pcDNA3.3. Plasmid inserts were excised by restriction enzyme digestion and used to template tail PCRs. The templates of human TRAIL and PTEN were obtained from our previously constructed expression vectors [19 (link), 47 (link)]. MEGAscript T7 kit (Ambion) was used to synthesize mRNA. However, m7GpppG was replaced with ARCA cap analog (New England Biolabs) and cytidine and uridine were replaced with 5-methylcytidine triphosphate and pseudouridine triphosphate (TriLink Biotechnologies) respectively. Reactions were incubated 5 h at 37°C followed by DNase treatment. Then, the reactions were treated with Antarctic Phosphatase (New England Biolabs) for 2 h at 37°C to remove residual 5′-triphosphates. The synthesized RNA was purified with Ambion MEGAclear spin columns (Ambion) and quantitated by Nanodrop (Thermo Scientific).