Journal of the American Chemical Society, 25 August, 2025, DOI：https://doi.org/10.1021/jacs.5c07380

Photoactivatable RNA Tags for Subcellular Photolabeling of RNA

Zhenyin Chen, Haodong Jiang, Lin Yuan, Tuoxin Yao, Xiaoxiao Rong, Wankai Gao, Chenhang Zeng, Liuqin He, Yulong Yin, Samie R. Jaffrey, Fangqing Zhao, Jinyang Zhang*, Pingyong Xu*, Xing Li*

Abstract

Photoactivatable fluorescent proteins enable precise analysis of subcellular protein dynamics, but a comparable approach for photoactivated tagging subcellular RNA remains lacking. Here, we develop PA-Broccoli, a photoactivatable fluorescent RNA tag that mimics photoactivatable green fluorescent proteins (PA-GFP). Similar to PA-GFP, PA-Broccoli is initially nonfluorescent but emits intense green fluorescence after ultraviolet light irradiation. Notably, complete photoactivation of PA-Broccoli results in a 6000-fold fluorescence enhancement with a rapid activation (t_1/2 = ～3 s), outperforming PA-GFP in sensitivity and speed by 1–2 orders of magnitude. Using PA-Broccoli, we first uncovered that cytoplasmic RNAs display restricted mobility compared to proteins. In addition, we revealed that circular RNAs undergo Ran-GTP-dependent nuclear export and slower cytoplasmic accumulation than linear mRNAs. Third, real-time imaging of mRNA within stress granules indicates that mRNA exchange throughout these granules is energy-dependent. These findings establish PA-Broccoli as a transformative tool for live-cell RNA tracking, offering unprecedented insights into RNA dynamics and regulatory mechanisms.

Article link：https://pubs.acs.org/doi/10.1021/jacs.5c07380