Nature-Inspired Chemical Probes for in-cell Labeling of Lipidized RNA and Identification of New Regulatory Enzymes

RNA modifications play pivotal roles in numerous cellular processes and human diseases. In addition to well-studied methyl-based modifications, hydrophobic prenyl-modifications have also been found in many RNA species. Here we report two chemical labeling strategies for tagging lipid-modified RNAs by taking advantage of a natural SelU-mediated tRNA geranylation process and the special reactivity of prenyl-groups. We synthesized a series of clickable geranyl-pyrophosphate analogs and identified two candidates for indirect RNA labeling using a two-step process, azidation-and-click-tagging of fluorescent dyes, namely ACT-Flu. We also developed a direct metabolic incorporation and biorthogonal tagging (MIBT-Tag) method based on the Ene-ligation of prenyl-groups. Both methods have been successfully applied to in-cell RNA labeling and the identification of new proteins associated with the geranylation process through proteomic and bioinformatic studies. These biochemical toolsets enable further in vivo applications to study prenylation pathways and monitor their status in both healthy and diseased cells.