Controlling In Vitro mRNA Polyadenylation by Monitoring Poly(A) Polymerase Consumption of ATP.

The poly(A) tail of mRNA plays a vital role in mRNA transcript stability, translational efficiency, and immunogenicity. Co-transcriptionally polyadenylated in vitro transcribed (IVT) mRNAs typically contain poly(A) tails of 50-120 nucleotide tail length due to limitations in production of template pDNA with longer poly(A) sequences. In contrast, post-transcriptional enzymatic polyadenylation of mRNA with poly(A) polymerase (PAP) presents a modular alternative to increase the tail length. However, the lack of real-time control strategies for PAP-mediated tailing has limited its broader applicability in mRNA production. Here, we develop a methodology for controlling poly(A) tail length in post-transcriptional polyadenylation of mRNA that uses adenosine triphosphate (ATP) consumption measured at-line to predict the poly(A) tail length. We establish a novel analytical method based on monolith reverse-phase chromatography to validate the poly(A) predictions. We were able to produce longer poly(A) tails and accurately determine their length in 300-700 nt range. The resulting longer poly(A) tailed reporter mRNAs outperformed the encoded and shorter poly(A) tailed mRNAs in cell-based assays. This work presents a new strategy for controlled post-transcriptional polyadenylation using ATP consumption as a process control metric, an approach which may in future be expanded to other NTP-dependent enzymatic conversions.