Characterization of nuclease stability and poly(A)-binding protein binding activity of chemically modified poly(A)...

The poly(A) tail plays a crucial role in mRNA stability and translation efficiency. Chemical modification of the poly(A) tail is a promising approach for stabilizing mRNA against deadenylation. In this study, we investigated the effect of poly(A) chemical modifications using phosphorothioate (PS), 2'-fluoro (2'-F), 2'-O-methyl (2'-OMe), and 2'-O-methoxyethyl (2'-MOE) modifications. Notably, PS, 2'-OMe, and 2'-MOE modifications conferred resistance to CAF1, an enzyme responsible for deadenylation. Interestingly, only the PS modification retained the poly(A)-binding protein (PABP) binding activity, which is critical for translation, whereas 2'-F, 2'-OMe, and 2'-MOE modifications abolished this activity. Beyond the PS modification, the combination of 2'-F, 2'-OMe, and 2'-MOE modifications resulted in enhanced resistance to both CAF1 and other nucleases. Based on these results, a 12-nucleotide unmodified poly(A) sequence was inserted upstream of the modified poly(A) to confer both nuclease resistance and PABP-binding activity. Notably, the resulting poly(A) formulation significantly prolonged protein expression in cultured cells and mouse skin when applied to epidermal growth factor-encoding therapeutic mRNA. Collectively, this study presents a design concept for poly(A) chemical modifications to achieve durable protein expression from mRNA, offering a promising strategy for enhancing the function of mRNA-based therapeutics.