Nucleoside modification and evolution of the wobble rule

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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.