Single-cell imaging of N4-acetylcytidine-modified RNA using fluorine metabolic labeling mediated proximity ligation assay.

N 4-Acetylcytidine (ac4C) is an emerging epitranscriptomic mark involved in regulating RNA stability, translation, and gene expression. Despite its emerging role in gene regulation and disease, current methods for in situ detection of ac4C-modified RNA lack sensitivity and specificity. To overcome these challenges, we developed fluorine metabolic labeling mediated proximity ligation assay (FMPLA), a method combining fluorine-based metabolic labeling with a proximity ligation assay for precise detection of newly synthesized fluoro-metabolically labeled ac4C sites. This approach enables high-sensitivity visualization of multiple RNA species, and provides insights into the abundance and spatial dynamics of ac4C-modified RNAs during the cell cycle and under chemotherapeutic stress. Additionally, FMPLA reveals distinct RNA modification patterns in drug-resistant cancer cells, highlighting its potential in studying functions and mechanisms of RNA ac4C modification.