Chemical pulldown of Prenylated-Adenosine-Incorporated RNA via sequential Fluorination-Azidation and a click reaction targeting prenyl functionality

Abstract

In recent years, modified nucleotides have attracted widespread attention in the field of chemical biology, biochemistry and beyond. Although some achievements have been made, such as high-throughput sequencing and biological roles, their molecular mechanism remains elusive. We report here a standard protocol for targeting prenyl-modified nucleic acids (i6A) using trimethylsilyl azide and Selectfluor as well as propargyl biotin (K3). After optimizing the conditions, we applied this method to fluorescent labeling and enrichment of i6A-RNA. Finally, we discussed the application scope of chemical pull-down assays in i6A-modified nucleic acids, including their use for detecting and identifying RNA-protein interactions, studying RNA metabolism, and studying gene expression regulation.