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

In addition to length, nucleotide compositions of A, C, G, and U may be an important factor at the 5' UTR. Generally, high GC content is commonly associated with increased structural complexity 27 . G can base pair with both C and U and induce the formation of more secondary structures 27 . The increase of U content may introduce additional chemically modified nucleotides into 5' UTR if modified U, such as pseudouridine are used in mRNA synthesis. To understand the effects of nucleotide compositions, we performed nucleotide compositions analysis and designed a series of 5' UTRs (NCA-1 to NCA-8). When adjusting nucleotide composition, the minimal numbers of G and C are 3 and 4, respectively, due to the transcription start site determined by the T7 promoter sequence (GG) and the use of Kozak sequence (GCCACC). These 5' UTRs were also built to avoid secondary structures (Supplementary Fig. 2b) , thereby minimizing the impact of structures on translation initiation. As shown in Fig. 3b , a decrease of relative luciferase activity was observed with less A and more G when the amount of C and U were constant (NCA-1 vs NCA-2; NCA-1 vs NCA-3). When G and U were minimal, the increase of C and decrease of A content showed a moderate decrease of luciferase expression (NCA-3 vs NCA-4). When holding G content to the minimal of 3, the inclusion of U boosted relative luciferase activity (NCA-5 vs NCA-6 vs NCA-7 vs NCA-8). Moreover, these 5' UTR with ACU outperformed those consisting of mostly AC and AG, suggesting the importance of U on 5' UTR. Consequently, two constructs with high U contents, NCA-7 and NCA-8, were selected for further studies.