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-oQQF 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.

A Kinetic and Fluorogenic Enhancement Strategy for Labeling of Nucleic Acids

“…To our knowledge, PINK is the first example of a fluorogenic intercalating agent exhibiting enhanced fluorescence upon covalently reacting with nucleic acids in living cells. Previous studies involving the chemical modification of cellular vinyl‐modified DNA by bioorthogonal methods have depended on cell fixation and denaturation to facilitate tetrazine‐alkene reactions [20, 30, 31, 54, 55] . The new capability of reacting bioorthogonal functional groups in native DNA and RNA will open up new opportunities in biological and translational research such as the real‐time study of chromosome segregation in animals, [56] and predicting nucleoside drug responses of cancer patients [14] …”

The unexplored potential of quinone methides in chemical biology

“…Furthermore, the authors also demonstrated metabolic incorporation of VTdT into endogenous double stranded DNA (dsDNA) to attain DNA live cell imaging by QM-mediated ligation. Gubu et al 22 (Figure 2). These experiments indicated that VTdT was incorporated into genomic DNA at both genomic and mitochondrial DNA level and subsequent biorthogonal ligation with oQQF afforded both nuclei and mitochondrial staining.…”

“…The application of QM-mediated ligations has also been exemplified for DNA labelling in living cells. 22 In vitro experiments revealed that a VT modified thymidine (VTdT) could be labelled with an oQQM precursor within 6 hours. Similarly, when VTdT was incorporated within a ssDNA synthetic oligonucleotide the ligation with oQQM modified fluorescein (oQQF) reached its plateau after 6 hours, but interestingly this was faster when the modified base was incorporated within synthetic dsDNA, as reaction completion was reached within 1h incubation for this substrate.…”

Covalent labeling of nucleic acids

“…15), which was used for DNA labeling in a hetero-Diels-Alder reaction with an o-quinoline quinone methide. 213 4.1.4 RNA labeling with alkyne-modified nucleosides. Similar to EdU, ethynyl uridine (EU) is the most common ribonucleoside used for metabolic labeling of RNA.…”