Semi-Enzymatic Synthesis of Pseudouridine

Modifications of RNA molecules have a significant effect on the structure and function of these RNAs. Many different examples of RNA modifications have been observed and each contribute in various forms to ensure proper biological activity. One of the most common modifications discovered is pseudouridine. The pseudouridine modification occurs in key dynamic locations in many RNA molecules. It is hypothesized that this modification plays an important structural role in the formation of a three-dimensional RNA structure, as well as assisting in the dynamics of that structure. Despite its prevalence in natural RNA sequences, organic and biological synthesis of pseudouridine has been difficult to perform at high yields. In this research, a new synthetic scheme is developed for the production of free pseudouridine in high yields by utilizing the reverse reaction of a pseudouridine glycosidase enzyme (YeiN). The cloning and overexpression of YeiN were successfully completed. Due to the COVID-19 outbreak, this project was halted before its completion. However, preliminary results show that this enzyme that typically participates in pseudouridine degradation can be used for the production of free pseudouridine in appreciable amounts. These results are promising for the synthesis of isotope-labeled pseudouridine for future structural and dynamics studies by NMR spectroscopy