The recent COVID-19 pandemics have demonstrated the great therapeutic potential of in vitro transcribed (IVT) mRNAs, but improvements in their biochemical properties, such as cellular stability, reactogenicity and translational activity, are critical for further practical applications in gene replacement therapy and anticancer immunotherapy. One of the strategies to overcome these limitations is the chemical modification of a unique mRNA 5′-end structure, the 5′-cap, which is responsible for regulating translation at multiple levels. This could be achieved by priming the in vitro transcription reaction with synthetic cap analogs. In this study, we combined a highly efficient trinucleotide IVT capping technology with several modifications of the 5′ cap triphosphate bridge to synthesize a series of 16 new cap analogs. We also combined these modifications with epigenetic marks (2′-O-methylation and m6Am) characteristic of mRNA 5′-ends in higher eukaryotes, which was not possible with dinucleotide caps. All analogs were compared for their effect on the interactions with eIF4E protein, IVT priming, susceptibility to decapping, and mRNA translation efficiency in model cell lines. The most promising α-phosphorothiolate modification was also evaluated in an in vivo mouse model. Unexpected differences between some of the analogs were analyzed using a protein cell extract pull-down assay.

中文翻译：

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具有三磷酸链修饰的三核苷酸帽类似物：作为 mRNA 加帽试剂的合成、性质和评估


最近的 COVID-19 大流行证明了体外转录 （IVT） mRNA 的巨大治疗潜力，但其生化特性的改进，如细胞稳定性、反应原性和翻译活性，对于基因替代疗法和抗癌免疫疗法的进一步实际应用至关重要。克服这些限制的策略之一是对独特的 mRNA 5′ 末端结构 5′ 帽进行化学修饰，该结构负责在多个水平上调节翻译。这可以通过用合成帽类似物引发体外转录反应来实现。在这项研究中，我们将高效的三核苷酸 IVT 加帽技术与 5' 帽三磷酸桥的几种修改相结合，合成了一系列 16 种新的帽类似物。我们还将这些修饰与高等真核生物中 mRNA 5'-末端特征的表观遗传标记 （2'-O-甲基化和 m6Am） 相结合，这在二核苷酸帽中是不可能的。比较所有类似物对模型细胞系中与 eIF4E 蛋白相互作用、 IVT 引发、脱帽敏感性和 mRNA 翻译效率的影响。最有前途的 α-硫代磷酸酯修饰也在体内小鼠模型中进行了评估。使用蛋白质细胞提取物 pull-down 测定法分析一些类似物之间的意外差异。