Nucleoside modification and evolution of the wobble rule

为了确定哪些氢键负责反密码子第一位的修饰尿苷与密码子第三位的鸟苷的结合，探索了将修饰鸟苷引入RNA分子特定位置的方法作为第一步。结果发现了一种酶促合成5 ′-磷酸6-硫代鸟苷的方法，并发现通过转录反应可以将肌苷和6-硫代鸟苷引入RNA分子中，进而探索了一种连接不同RNA分子制备可翻译mRNA分子的方法。使用T4 DNA和RNA连接酶的常规方法在产率和特异性方面存在问题。最近报道的催化双链RNA连接的T4 RNA连接酶2可用于连接，但产物不能在来自大肠杆菌的无细胞系统中翻译。这可能是由于酶的某些未知活性。另一方面，来自小麦胚的RNA连接酶可用于以高产量连接RNA片段。然而，很难检查如果另一种酶工作良好，可以除去的2 ′-磷酸是否从连接产物中有效除去。在研究过程中，一个竞争研究小组报道了修饰的尿苷和鸟苷之间摆动碱基对的X射线晶体学结构。结构看起来与该项目工作假设中假设的氢键模式不一致。然而，我们认为，晶体中观察到的碱基对可能仅在结晶条件下最稳定，其中pH低于生理条件。因此，发表了一份手稿，其中详细描述了我们假设的物理化学和生物化学背景。

In order to determine which hydrogen bonds are responsible for the binding of modified uridines at the first position of the anticodon to the guanosine at the third position of the codon, a method for introducing modified guanosines into a specific position of RNA molecules was explored as a first step. As a result, a method for enzymatic synthesis of 6-thioguanosine 5'-monophosphate was found. It was also found that inosine and 6-thioguanosine can be introduced into RNA molecules by transcription reactions.Then, a method for ligation of different RNA molecules for the preparation of translatable mRNA molecules was explored. Conventional methods with T4 DNA and RNA ligases had problems in yields and specificity. T4 RNA ligase 2, which was reported recently to catalyze ligation of double-stranded RNA, was usable for the ligation, but the products were not translated in a cell-free system from Escherichia coli. This may have been due to some unknown activity of the enzyme. On the other hand, an RNA ligase from wheat embryos was useful for linking RNA fragments in high yields. However, it was difficult to check if the 2'-phosphate, which could be removed if another enzyme worked well, was efficiently removed from the ligation product. This problem should be solved in future researches.In the course of the research, an X-ray crystallographic structure of the wobble base pair between a modified uridine and the guanosine was reported from a competing research group. The structure looked inconsistent with the hydrogen bonding pattern postulated in the working hypothesis of this project. However, it was possible, in our opinion, that the base pair observed in the crystal was most stable only under the condition of the crystallization, where the pH is lower than the physiological condition. Therefore, a manuscript was published, in which the physicochemical and biochemical backgrounds of our hypothesis were described thoroughly.