The Effect of Cap Structure and Poly(A) Positioning on mRNA Translation Efficiency

We investigated the relationship between mRNA structure and translation activity using our recently developed chemical method to add a cap structure to an oligonucleotide and a technique for purifying capped mRNAs via reversed‐phase high‐performance liquid chromatography (HPLC) employing a photoreactive tag. Specifically, we designed and synthesized mRNA constructs in which the typical eukaryotic elements—the 5′ cap and the 3′ poly(A) tail—were interchanged in position. We examined how this inversion affected translation efficiency. The results revealed that this reversed configuration abolished the synergistic enhancement of translation typically observed with correctly positioned cap and poly(A) tail, resulting in a substantial decrease in activity compared to the native orientation. Interestingly, focusing solely on the cap structure, capping at the 3′ end—though less effective than at the 5′ end—still promoted a measurable increase in translation. Moreover, the poly(A) sequence placed at the 5′ end was found to suppress translation. We also prepared mRNAs containing a poly(A) tail with reversed 3′–5′ orientation and demonstrated that this inverted poly(A) could still enhance protein synthesis.