信使RNA（mRNA）是一类细胞内作为遗传信息载体的单链核酸大分子。由于mRNA在生产上的快速性、在细胞内的安全性和在翻译时的高效性等优势，目前mRNA以及被广泛应用于医药健康领域，譬如传染病预防治疗、肿瘤免疫治疗、蛋白替代疗法、基因编辑等。而mRNA天然结构上的不稳定性导致其在生产制造、储藏运输、药物应用等场合均存在易降解的风险。这一特性大大限制了mRNA的应用空间并提高了mRNA的生产成本。为了解决这一难题，本论文从mRNA的修饰、结构以及序列三个角度出发，探究提高mRNA稳定性并使其在细胞内稳定表达的相关策略。在mRNA的修饰方面，从mRNA的5’端帽结构修饰出发，选择了多种在细胞环境内具有活性的不同病毒来源的mRNA加帽酶进行体外加帽活性的测试。通过大肠杆菌蛋白表达体系BL21（DE3）和促溶标签麦芽糖结合蛋白（MBP）成功实现了这些mRNA加帽酶的异源表达。基于在HEK293T细胞内的蛋白表达情况，蓝舌病毒加帽酶被证明比通用牛痘病毒加帽酶的活性高出38%。在mRNA的结构方面，从mRNA的体外环化方式出发，在筛选对比T4 DNA连接酶、T4 RNA连接酶1和T4 RNA连接酶2的基础上，设计了20 bp的同源臂序列提高酶促连接方式的环化效率。同时也筛选出来源于T4噬菌体胸苷酸核酶（Td）基因的组I内含子自剪接序列作为综合环化效率较高的核酶序列。在对比几种环化方式之后，确定基于T4 Rnl 2的酶促连接方式为效率最高的环化方式。在mRNA的序列方面，从内部核糖体进入位点（IRES）出发，基于不同环化方式、不同细胞环境的实验结果，筛选出来源于青蟹双顺反子病毒（MCDV）的IRES序列是一种具有较高的核糖体结合能力并且适应不同的细胞环境可以广泛应用在环状mRNA中的IRES序列。此外，5’端帽结构和IRES结构的协同效应被证明能够显著提升线性mRNA在细胞内的蛋白表达量。最终，本论文通过多种策略提高了mRNA的稳定性，并构建了两种在细胞内能够稳定并高效表达的mRNA载体。一种是通过蓝舌病毒加帽酶加帽并添加IRES序列的线性mRNA载体；另一种是通过T4 RNA连接酶2环化并融合MCDV IRES的circmRNA载体。这些更为稳定的mRNA载体的构建可适用于多种核酸疗法的应用场景，从而推动mRNA产业的发展并扩大其应用的空间。
Messenger RNA (mRNA) is a kind of single-stranded nucleic acid macromolecule that serves as carriers of genetic information in cells. Due to the advantages of rapid production, intracellular safety and high efficiency in translation, mRNA is now widely used in medicine and health fields, such as infection prevention and treatment, tumor immunotherapy, protein replacement therapy and gene editing. However, the natural structural instability of mRNA leads to the risk of degradation in manufacturing, storage, transportation, and drug application. This property greatly limits the application space and increases the cost of mRNA production. To address this challenge, this article explored strategies to improve the stability and intracellular expression of mRNAs from three perspectives: mRNA modification, structure and sequence. In terms of mRNA modification, a variety of mRNA capping enzymes of different viral origins, which were active in the cellular environment, were selected for experiment in vitro. Heterologous expression of these mRNA capping enzymes was successfully achieved by the E. coli BL21 (DE3) and the maltose binding protein (MBP). Based on protein expression results in HEK293T cells, bluetongue virus capping enzyme was shown to be 38% more active than generic vaccinia capping enzyme. In terms of mRNA structure, a 20 bp homologous arm sequence was designed to improve the cyclization efficiency of the enzymatic ligation method based on screening comparing T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl 1) and T4 RNA ligase 2 (T4 Rnl 2). Meanwhile, the group I intron self-splicing sequence derived from the T4 phage thymidylate synthase (Td) gene was also screened as the ribozyme sequence with higher cyclization efficiency. After comparing several mRNA ligation methods, the enzymatic ligation method based on T4 Rnl 2 was identified as the most efficient cyclization method. In terms of mRNA sequences, based on the internal ribosome entry site (IRES), the IRES sequence from mud crab dicistrovirus (MCDV) was selected as the IRES sequence with high ribosome binding ability and adaptable to different cellular environments, which meant MCDV IRES could be widely used in circular mRNAs (circmRNAs). Furthermore, the synergistic effect of the 5‘ end-cap structure and the IRES structure was shown to significantly enhance the protein expression of linear mRNAs in cells. Ultimately, these results had improved the stability of mRNA through multiple strategies and constructed two mRNA vectors that can be stably and efficiently expressed in cells. One is a linear mRNA vector capped by bluetongue virus capping enzyme and added with IRES sequence. The other is a circmRNA vector cyclized by T4 RNA ligase 2 and added with MCDV IRES. The construction of these more stable mRNA vectors could be applied to a variety of nucleic acid therapy application scenarios, thus promoting the development of mRNA industry and expanding its application space.
