Selection of Short 5′-UTR of Chemically Synthesized mRNA to Improve Translation Efficiency

The advent of mRNA medicine, initially implemented as a vaccine during the coronavirus disease 2019 (COVID-19) pandemic, has attracted interest in diverse therapeutic applications, including cancer vaccines and protein replacement therapies. Our group recently established a method for the complete chemical synthesis of mRNA. Although this approach has some advantages, chemically synthesized mRNA is limited to approximately 150 nucleotides in length and necessitates optimized designs for untranslated regions (UTRs) and coding sequences. To address this challenge, we investigated whether the non-reporter-based selection methods, including ribosome profiling and polysome profiling, which were often used for UTR optimization in long mRNA, could be adapted for short mRNA to identify highly translated UTR sequences. Using these methods, we collected mRNAs that interacted with ribosomes and analyzed their 5'-UTR sequences. We successfully identified a 9-nucleotide 5'-UTR that demonstrated approximately double the translation efficiency of the Kozak sequence, a widely used positive control. This work highlights the adaptability of ribosome-focused selection techniques for short, chemically synthesized mRNA and provides a foundation for effective sequence design. These findings advance the development of chemically synthesized mRNA as a viable alternative to in vitro-transcribed mRNA, paving the way for innovative therapeutic applications.