Live‐Cell RNA Imaging via Clickable Tri PPP ro Nucleotide Reporters

Abstract Understanding RNA synthesis and dynamics in cells requires efficient labeling strategies that are not only compatible with cellular environments but can be performed in living cells. We developed a robust, bio‐orthogonal approach for live‐cell RNA labeling using Tri PPP ro (triphosphate prodrug) chemistry. This strategy enables the intracellular delivery of sterically demanding nucleoside triphosphates modified with inverse electron‐demand Diels–Alder (IEDDA)‐reactive groups, specifically trans ‐cyclooctene (2TCO a ) and bicyclo[6.1.0]nonyne (BCN). Once hydrolyzed inside cells, these Tri PPP ro‐modified uridines and cytidines are metabolically incorporated into nascent RNA by endogenous RNA polymerases. Subsequent IEDDA reaction with a dual‐fluorogenic tetrazine‐cyanine styryl dye conjugate allows wash‐free, high‐contrast imaging of RNA synthesis in cells. We demonstrate efficient RNA labeling, including nucleolar localization and specificity for newly transcribed RNA, validated by transcriptional inhibition and colocalization with ribosomal RNA. Comparative analyses confirm that Tri PPP ro delivery surpasses conventional transporter‐based systems in both labeling efficiency and cellular compatibility. This platform offers a modular, non‐genetic, and highly specific method for real‐time RNA imaging, with broad applicability for RNA biology and antiviral research.