Bioorthogonal Metabolic DNA Labelling using Vinyl Thioether‐Modified Thymidine and o‐Quinolinone Quinone Methide

Abstract Bioorthogonal metabolic DNA labeling with fluorochromes is a powerful strategy to visualize DNA molecules and their functions. Here, we report the development of a new DNA metabolic labeling strategy enabled by the catalyst‐free bioorthogonal ligation using vinyl thioether modified thymidine and o ‐quinolinone quinone methide. With the newly designed vinyl thioether‐modified thymidine (VTdT), we added labeling tags on cellular DNA, which could further be linked to fluorochromes in cells. Therefore, we successfully visualized the DNA localization within cells as well as single DNA molecules without other staining reagents. In addition, we further characterized this bioorthogonal DNA metabolic labeling using DNase I digestion, MS characterization of VTdT as well as VTdT‐ o QQF conjugate in cell nuclei or mitochondria. This technique provides a powerful strategy to study DNA in cells, which paves the way to achieve future spatiotemporal deciphering of DNA synthesis and functions.