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Keywords:

  • bioorthogonal reporters;
  • Diels–Alder cycloaddition;
  • fluorescent probes;
  • metabolic labeling;
  • nucleosides

Abstract

5-Vinyl-2′-deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be visualized by using an inverse electron demand Diels–Alder reaction with a fluorescent tetrazine. VdU is incorporated by endogenous enzymes into the genomes of replicating cells, where it exhibits reduced genotoxicity compared to 5-ethynyl-2′-deoxyuridine (EdU). The VdU–tetrazine ligation reaction is rapid (k≈0.02 M−1 s−1) and chemically orthogonal to the alkyne–azide “click” reaction of EdU-modified DNA. Alkene–tetrazine ligation reactions provide the first alternative to azide–alkyne click reactions for the bioorthogonal chemical labeling of nucleic acids in cells and facilitate time-resolved, multicolor labeling of DNA synthesis.