Visualizing Newly Synthesized RNA by Bioorthogonal Labeling-Primed DNA Amplification

Monitoring RNA synthesis and spatial distribution can help to understand its role in physiology and diseases. However, visualizing newly synthesized RNA in single cells remains a great challenge. Here, we developed a bioorthogonal labeling-primed DNA amplification strategy to visualize newly synthesized RNA in single cells. The new bioorthogonal N6-allyladenosine nucleoside was prepared to metabolically label cellular newly synthesized RNAs. These allyl-functionalized RNAs then reacted with tetrazine-modified primers. These primers could initiate rolling circle amplification, producing tandem periodic long single DNA strands to capture hundreds of fluorescence probes for signal amplification. Using this method, we explored the subcellular distributions of newly synthesized RNAs. And we found that newly synthesized RNAs are spatially organized in a cell type-specific style with cell-to-cell heterogeneity.