Nature‐Inspired Chemical Probes for Labeling tRNAs Containing 2‐Thiouridine (s ² U)

RNA modifications play pivotal roles in numerous cellular processes and human diseases. In addition to the well-studied methyl-based modifications, hydrophobic prenyl-modifications have also been found in many RNA species. Herein, a chemical labeling strategy for tagging specific tRNAs by taking advantage of a natural SelU-mediated geranylation process is reported. A series of "clickable" geranyl-pyrophosphate analogs is synthesized and two candidates to label 2-thiouridine (s²U) containing tRNA using a two-step process, azidation-and-click-tagging with fluorescent dyes, namely ACT-Flu are identified. This method has also been applied to in-cell RNA imaging, providing a useful biochemical toolset to further study the biological functions of s²U-tRNAs and monitor their status in both healthy and diseased cells, since human tRNAs do not have the prenyl group on natural s²U34 modification.