PolyA tail segmentation improves the stability of the template DNA and increases the translatability of <i>in vitro</i> transcribed mRNA

PolyA tail regulates messenger RNA (mRNA) localization, stability, and translation. PolyA length affects the durability and translational activity of both endogenously and exogenously delivered mRNAs. However, long polyA stretches can undergo recombination during amplification in bacterial plasmids, impairing the production of in vitro transcribed mRNA with long polyAs. PolyA tail segmentation with heteronucleotide spacers has recently emerged as a solution. Here, we developed segmented polyA patterns that stabilize the sequence during DNA amplification and enhance mRNA translation. We designed 15 novel genetically modified polyA variants, differing in the length, placement, and frequency of spacers, and the overall length (from ~120 to 200 nucleotides). We evaluated their stability in DNA plasmids and homogeneity, translational activity, and durability in cell culture of the resulting mRNAs, comparing them to A90 tail and other known solutions, including those from existing mRNA vaccines. Selected sequences were validated in vivo. Surprisingly, we found that even frequent heteronucleotide insertions produce functional polyA tails. The most notable enhancements in protein production were observed for a segmented tail exceeding 200 nt in length [A30(CA15)11; up to six-fold compared to mRNA with A90 tails]. Our findings extend the scope of possible polyA modification strategies, offering new possibilities for advancing mRNA therapeutics.