Incorporation of Pseudouridine into RNA for Biochemical and Biophysical Studies

Abstract

Modification of RNA molecules has a significant effect on their structure and function. Many different examples of RNA modifications have been observed and each contributes in various ways to ensure proper biological activity. One of the most common modifications is pseudouridine which occurs in key dynamic locations in many RNAs. Despite its prevalence in natural RNA sequences, organic synthesis of pseudouridine has been challenging because of the stereochemistry requirement and the sensitivity of reaction steps to moisture. Herein, we describe the semi-enzymatic synthetic route for the synthesis of pseudouridine using adenosine-5’-monophosphate and uracil as the starting materials and a reverse reaction catalyzed by a pseudouridine monophosphate glycosidase. Moreover, we describe the conversion from nucleoside (pseudouridine) to nucleotide triphosphate (pseudouridine-5’-triphosphate) to incorporate into RNA via in vitro transcription for biochemical and biophysical studies.