Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo

Chunxi Zeng; Xucheng Hou; Jingyue Yan; Chengxiang Zhang; Wenqing Li; Weiyu Zhao; Shi Du; Yizhou Dong

Besides utilizing UTRs derived from endogenous genes, one alternative approach is to design mRNA UTRs via a de novo method, which is widely applied in protein engineering 24 . During the process of de novo design for UTRs, several general principles can be considered. For example, the Kozak sequence (GCCACC) located directly upstream of the translation start codon (AUG) can be included because it is a conserved sequence among many mammalian mRNAs for accurate start codon recognition 25 . Second, AUG within 5' UTR is excluded to avoid alternative translation initiation and mutation of the amino acid sequence 10 . Third, secondary structures may be minimized to reduce the energy barriers for smooth scanning of the translation initiation complex along the 5' UTR 13, 26 . Based on the knowledge in the literature, we designed 5' UTR of different length: 10nt, 30, 50nt, 70nt, and 90nt with minimal secondary structures as modeled by RNAfold, a wellestablished computational tool for the prediction of RNA secondary structures ( Supplementary Fig. 2a and Supplementary Table 2 ). We then prepared the mRNAs and evaluated their luciferase expression as mentioned above. The results showed that relative luciferase activity increased with 5' UTR length and peaked at 70nt (Fig. 3a) . Further increase of the length lowered luciferase expression. Therefore, 70nt was used as the optimal length for the subsequent design of 5'UTRs.