Chen Zhenkuan, Sun Zhaowei, Cheng Jiaqi, Ren Shujuan, He Qian, Xu Xue, Ma Qiang, Weng Haibo
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China.
School of Life Sciences, Zhengzhou University, Zhengzhou, Henan 450001, China.
Int J Biol Macromol. 2025 Sep;321(Pt 1):146218. doi: 10.1016/j.ijbiomac.2025.146218. Epub 2025 Jul 21.
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.
体外mRNA转录需要5'帽和3'聚腺苷酸尾才能在真核细胞中实现蛋白质表达，这些特征对mRNA疫苗和基因治疗的成功至关重要。然而，这些修饰增加了工业规模生产的复杂性和成本。我们在此开发了一种新型RNA构建体，用来自脑心肌炎病毒（EMCV）的内部核糖体进入位点（IRES）取代5'帽和非翻译区（UTR），并用复制依赖性组蛋白茎环（SL）结构取代3'聚腺苷酸尾。通过对各种3'UTR和SL结构的系统筛选，我们确定β-珠蛋白3'UTR与人类组蛋白SL结合是高效翻译的最佳配置。所得的mRNA构建体实现了与传统加帽和聚腺苷酸化mRNA相当的表达效率和稳定性（p>0.05），而无需帽或聚腺苷酸尾。这个创新系统为mRNA生产提供了一个简化的、具有成本效益的平台，同时为mRNA疫苗和基因治疗的未来应用提供了有前景的技术进步。
