As mRNA technology emerges as a groundbreaking innovation, an increasing number of studies are attempting to increase expression levels by optimizing the structure of mRNA, such as the 5' or 3' untranslated regions (UTR). We designed a novel loop structure in the poly(A) tail of an mRNA platform to improve the stability and duration. Protein expression analyses were conducted using bioluminescence, both in vitro and in vivo. Additionally, cellular and humoral responses to different antigens were confirmed using flow cytometry and ELISA of splenocytes and serum isolated from mice immunized with mRNA containing different poly(A) tail structures. The poly(A) tail with a loop structure exhibited higher bioluminescence signals, both in vitro and in vivo, and increased human erythropoietin (hEPO) expression in vivo compared to the other poly(A) tail groups. This indicates that the addition of a loop structure to the poly(A) tail region improves mRNA stability and efficiency. The results of analyzing cellular and humoral immunity by expressing HPV E6 and E7 antigens, and influenza virus HA antigens in mRNA with various poly(A) structures showed no significant difference in T cell immunity and antibody titer. In conclusion, the addition of stable structures, such as a loop to the poly(A) tail, can significantly increase the expression efficiency and stability of mRNA, but the expression difference did not have as much of an impact on the immune response as expected.
