While mRNA therapeutics developed in recent years show tremendous potential, there are still technical obstacles to overcome, two of the most significant being mRNA’s intrinsic instability and its immunogenicity. In order to address these challenges, several different approaches can be used, including optimization of 3 poly(A) tail length. mRNA is commonly polyadenylated co transcriptionally, by transcription from DNA template encoding the tail. However, long (>120 nt) stretches of thymidines in DNA sequence can lead to cloning difficulties, plasmid instability during culture and formation of multimeric plasmid structures. Enzymatic post-transcriptional addition of poly(A) tail is thus an attractive alternative. Very limited knowledge base is available on post-transcriptional polyadenylation (PA), therefore our main focus was to improve the understanding of in vitro PA reaction.
