Poly(A) variants supporting robust transmission stability in bacteria and high protein expression in animals for mRNA in vitro transcription

Poly(A) tail is crucial in regulating mRNA stability and protein translation. Thus, it is an essential element of mRNAs transcribed <i>in vitro</i> for mRNA medicines. However, the repetitive nature of the poly(A) tail can lead to significant poly(A) length variation and compromise the quality of the mRNA drug substance. Previous studies improved poly(A) transmission stability by inserting a non-adenosine spacer. Here, we designed new segmented poly(A) variants and evaluated their transmission stability in bacteria transformation and their ability in supporting protein expression in animals. We identified specific variants with multiple non-adenosine insertions that can maintain high transmission stability with robustness in <i>Escherichia coli</i> and facilitate high protein expression in animals. Among the newly designed poly(A) variants, RG2 showed high and consistent transmission stability comparable to the A30-70 variant that is the industry gold standard but higher protein expression in animals than A30-70. We also isolate new factors that can influence the stable transmission of poly(A), such as poly(A) surrounding sequences and bacteria culture temperature. Thus, our work offers new tools valuable for rapidly developing mRNA vaccines and therapeutics.