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

To screen endogenous UTRs in mammalian cells, we utilized a bioinformatics analysis of global gene expression as a starting point. Previously, the average half-life (h) and translation rate constant [protein copy number/(mRNA×h)] of 4248 mRNAs and corresponding proteins were quantified in mammalian cells 19 . According to their data, we calculated the translation capacity per mRNA molecule using the following equation: average half-life (h) × translation rate constant [protein copy number/(mRNA×h)] × protein length (amino acids) = translation capacity per mRNA molecule (total amino acids/mRNA) (Supplementary Table 1 ). These results indicated the number of amino acids produced by a single mRNA during its half-life. The mRNA from the murine Rps27a gene was found to possess the highest translation capacity per mRNA molecule. Rps27a is a housekeeping gene that encodes one of the components of the ribosome 40S subunit 20 . Two proteincoding transcripts exist for the murine Rps27a gene (named S27a-44 and S27a-45). These two transcripts have different 5' UTRs and share the same 3' UTR (Supplementary Table 2 and Supplementary Table 3 ). Therefore, we generated two mRNAs utilizing S27a-45 and S27a-44 5' UTRs, respectively. In these two 5' UTRs, we found putative terminal oligo-pyrimidine (TOP) motifs consisting of 4-15 clustered pyrimidines (C and U), which were reported to negatively regulate mRNA translation (Supplementary Table 2) 21 . Subsequently, we removed the TOP motifs from S27a-44 and S27a-45 5' UTRs and constructed two additional 5' UTRs: S27a-44' and S27a-45'. The mRNAs encoding Firefly luciferase were synthesized with these 5' UTRs and were delivered to Hep3B and 293T cells using lipofectamine 3000. The control UTRs include CYBA UTRs (named CYBA) 12 , alpha globin UTRs (named AG) 13 , and modified alpha globin UTRs (AG+G with a complete Kozak sequence and 5AG+G without 3' UTR) 18 . 5' UTR from S27a-44 was better than that from S27a-45 (Fig. 2a) . Removal of the putative TOP motif from S27a-45 moderately improved expression, while S27a-44' without the TOP motif increased the relative luciferase activity over 70% compared to the S27a-44 in the two cell lines. S27a-44' was comparable to CYBA and AG+G, the two best controls (Fig. 2a) .