mRNA Synthesis and Optional Post-Transcriptional Modifications
mRNA synthesis:In vitro transcription (IVT) involves the synthesis of RNA from a linear DNA template, such as linearized plasmid DNA, containing an appropriate transcription promoter. mRNA is typically enzymatically synthesized by T7 RNA polymerase using nucleotide triphosphate (NTP) building blocks. Incorporating modified nucleotides such as N1-Methylpseudouridines can help to reduce immunogenicity and enhance translation efficiency.
mRNA modifications:mRNA 5’ capping can be performed co-transcriptionally by using cap analogs in the mRNA synthesis mixture, or post-transcriptionally by using specific capping enzymes. In case that the DNA template does not code for a 3’ polyA tail, it can be introduced enzymatically by using PolyA polymerase after the mRNA synthesis. These modifications enhance protein translation, improve stability, and reduce immunogenicity as required.
Next Step:Watch the mRNA synthesis video
