Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA body and polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases-SP6, T7 and polyA polymerase. To verify the efficiency of incorporation of ATP analogues in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'-mononucleotides. The method also estimated the average polyA tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the polyA tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.

中文翻译：

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mRNA polyA尾中的磷酸二酯修饰可防止去腺苷酸化而不影响蛋白质表达

化学修饰能够制备具有增强稳定性和翻译活性的 mRNA。在这项研究中，我们探讨了 mRNA 体和 polyA 尾中 5',3'-磷酸二酯键的化学修饰如何影响真核 mRNA 的生物学特性。为了获得修饰和未修饰的体外转录 mRNA，我们使用了 ATP 和 ATP 类似物在 α-磷酸修饰（包含 O-to-S 或 O-to-BH3 取代）和三种不同的 RNA 聚合酶——SP6、T7 和 polyA 聚合酶. 为了验证在 ATP 存在下掺入 ATP 类似物的效率，我们开发了一种液相色谱 - 串联质谱 (LC-MS/MS) 方法，用于基于转录物彻底降解为 5'-单核苷酸的修饰频率的定量评估. 该方法还估计了平均 polyA 尾长，从而为建立 mRNA 的结构-生物学特性关系提供了一种通用工具。我们发现，与未修饰的 mRNA 相比，polyA 尾部含有硫代磷酸酯基团的 mRNA 更不易被 3'-脱腺苷酸酶降解，并且在培养细胞中有效表达，这使它们成为有用的研究工具和未来基于 mRNA 的疗法开发的潜在候选者.