Rational design of a cost-effective IRES-stem-loop translation system independent of cap and poly(A) tail

In vitro mRNA transcription requires a 5' cap and a 3' poly(A) tail to enable protein expression in eukaryotic cells-features critical to the success of mRNA vaccines and gene therapies. However, these modifications add complexity and cost to industrial-scale production. We here developed a novel RNA construct that replaces the 5' cap and untranslated region (UTR) with the internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV), and substitutes the 3' poly(A) tail with a replication-dependent histone stem-loop (SL) structure. Through systematic screening of various 3' UTRs and SL structures, we identified the β-globin 3' UTR combined with the Homo sapiens histone SL as the optimal configuration for efficient translation. The resulting mRNA construct achieved comparable expression efficiency and stability to conventional capped and polyadenylated mRNAs (p > 0.05), without requiring either a cap or poly(A) tail. This innovative system offers a streamlined, cost-effective platform for mRNA production, while offering a promising technological advancement for future applications in mRNA vaccines and gene therapy.