# Live‐Cell RNA Imaging via Clickable TriPPPro Nucleotide Reporters

## TL;DR

A new method for live-cell RNA imaging uses TriPPPro chemistry to label RNA in real time with high contrast and specificity.

## Contribution

A bio-orthogonal TriPPPro strategy enables efficient, wash-free live-cell RNA labeling with IEDDA-reactive nucleosides.

## Key findings

- TriPPPro delivery outperforms conventional systems in RNA labeling efficiency and cellular compatibility.
- The method allows high-contrast imaging of newly transcribed RNA, including nucleolar localization.
- Labeling specificity was validated using transcriptional inhibition and ribosomal RNA colocalization.

## 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 TriPPPro (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 (2TCOa) and bicyclo[6.1.0]nonyne (BCN). Once hydrolyzed inside cells, these TriPPPro‐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 TriPPPro 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.

The intracellular delivery of trans‐cyclooctene and bicyclo[6.1.0]nonyne as sterically demanding groups for the inverse electron‐demand Diels–Alder was achieved by the triphosphate prodrug (TriPPPro) strategy. Once hydrolyzed inside cells, the modified uridines and cytidines are metabolically incorporated into nascent RNA, are labelled by a dual‐fluorogenic tetrazine‐dye to allow wash‐free, high‐contrast imaging of RNA synthesis in cells.