Imaging Low-Abundance, Short Cellular RNAs Using G-quadruplex FISH

Visualizing cellular RNAs enables spatial resolution at the single-cell level. Fluorescence in situ hybridization (FISH) is a facile tool; however, it is not sensitive enough to detect low-abundance or short RNAs in cells due to limited signal gain. Here, we present G-quadruplex FISH, a sensitive RNA imaging method that synergizes G-quadruplex peroxidase-driven tyramide deposition with proximity labeling chemistry. It eliminates nucleic acid amplification yet achieves efficient signal amplification through catalytic tyramide polymerization. Compared to conventional FISH, G-quadruplex FISH reduces background and enhances signal-to-background ratio, enabling the detection of RNAs down to 25.7 copies/cell. Its high-gain imaging enables the visualization of short RNAs in cells. Using G-quadruplex FISH, we explored the immunotoxicity of endotoxins and screened its antagonists. G-quadruplex FISH addresses FISH's sensitivity limitation, facilitating investigation of the roles of different RNAs involved in cellular function and disease.