Nature‐Inspired Chemical Probes for Labeling tRNAs Containing 2‐Thiouridine (s²U)

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 (s 2 U) containing tRNA using a two‐step process, a zidation‐and‐ c lick‐ t agging 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 s 2 U‐tRNAs and monitor their status in both healthy and diseased cells, since human tRNAs do not have the prenyl group on natural s 2 U34 modification.