Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides

Abstract

Commonly used metabolic labels for DNA, including 5-ethynyl-2'-deoxyuridine (EdU) and BrdU, are toxic antimetabolites that cause DNA instability, necrosis, and cell-cycle arrest. In addition to perturbing biological function, these properties can prevent metabolic labeling studies where subsequent tissue survival is needed. To bypass the metabolic pathways responsible for toxicity, while maintaining the ability to be metabolically incorporated into DNA, we synthesized and evaluated a small family of arabinofuranosyl-ethynyluracil derivatives. Among these, (2'S)-2'-deoxy-2'-fluoro-5-ethynyluridine (F-ara-EdU) exhibited selective DNA labeling, yet had a minimal impact on genome function in diverse tissue types. Metabolic incorporation of F-ara-EdU into DNA was readily detectable using copper(I)-catalyzed azide-alkyne "click" reactions with fluorescent azides. F-ara-EdU is less toxic than both BrdU and EdU, and it can be detected with greater sensitivity in experiments where long-term cell survival and/or deep-tissue imaging are desired. In contrast to previously reported 2'-arabino modified nucleosides and EdU, F-ara-EdU causes little or no cellular arrest or DNA synthesis inhibition. F-ara-EdU is therefore ideally suited for pulse-chase experiments aimed at "birth dating" DNA in vivo. As a demonstration, Zebrafish embryos were microinjected with F-ara-EdU at the one-cell stage and chased by BrdU at 10 h after fertilization. Following 3 d of development, complex patterns of quiescent/senescent cells containing only F-ara-EdU were observed in larvae along the dorsal side of the notochord and epithelia. Arabinosyl nucleoside derivatives therefore provide unique and effective means to introduce bioorthogonal functional groups into DNA for diverse applications in basic research, biotechnology, and drug discovery.

Fig. 1.

Arabinofuranosyl-5-ethynyluridine derivatives, acute toxicity, and metabolic labeling. (A) Structures of synthetic nucleosides studied in this report. (B) HeLa cell metabolism according to the Alamar Blue assay after 72 h in DMEM + 10% FCS solutions containing variable concentrations of each nucleoside. See SI Appendix, Fig. S2 for cellular proliferation assays. (C) Metabolic labeling of genomic DNA in HeLa cells following a 24 h incubation with 10 μM of each nucleoside. After washing and fixing the cells, ethynyl-modified DNA was stained using AlexaFluor 488 azide and Cu(I), and total cellular DNA was stained with DAPI. Negative controls received identical treatments, but were not exposed to a synthetic nucleoside. (Scale bar: 50 μm.)

Fig. 2.

Comparison of EdU and F-ara-EdU for metabolic labeling of DNA. HeLa cells were treated with variable concentrations of EdU or F-ara-EdU for 72 h. After washing and fixing the cells, ethynyl-modified DNA was stained using AlexaFluor 488 azide and Cu(I), and total DNA was stained with DAPI. (Scale bar: 50 μm.) See SI Appendix, Figs. S6 and S7 for quantitative analyses by FACS at 24 and 72 h.

Fig. 3.

Pulse-chase labeling of DNA in HeLa cells with EdU or F-ara-EdU (1 μM) for 72 h, followed by a BrdU chase (10 μM) for 24 h. Following fixation, ethynyl-modified DNA was stained using AlexaFluor 488 azide and Cu(I), BrdU was stained with BrdU antibody-AlexaFluor 647 conjugate, and total DNA was stained with DAPI. (Scale bar: 100 μm.) See SI Appendix, Fig. S10 for quantitative analyses by FACS.

Fig. 4.

Pulse labeling of DNA in HeLa cells with F-ara-EdU (10 μM), BrdU (10 μM), or a 1∶1 mixture (10 μM each) followed by a cold chase. HeLa cells were incubated with F-ara-EdU and/or BrdU for 24 h, washed, and grown in rich media lacking synthetic nucleosides for 5 d and fixed. Ethynyl-modified DNA was stained using AlexaFluor 488 azide and Cu(I), BrdU was stained with BrdU antibody-AlexaFluor 647 conjugate, and total DNA was stained with DAPI. (Scale bar: 10 μm.) See SI Appendix, Figs. S13 and S14 for variable-magnification images.

Fig. 5.

Pulse-chase labeling of Zebrafish embryos using F-ara-EdU and BrdU. (A) Embryos microinjected with F-ara-EdU at 0 hpf and with BrdU at 5 hpf. (B) Embryos microinjected with F-ara-EdU at 0 hpf and with BrdU at 10 hpf. (C) Embryos microinjected with EdU at 0 hpf and with BrdU at 10 hpf. Negative controls (D) remained untreated. Larvae were allowed to develop until 3 d after fertilization, fixed, and stained using AlexaFluor 488 azide and Cu(I), and BrdU antibody-AlexaFluor 647 conjugate. Pictures show lateral views of tail regions. The white arrows indicate cells surrounding the notochord labeled with only F-ara-EdU; arrowheads indicate cells associated with epithelia labeled with only F-ara-EdU. Abbreviations: dor, dorsal; cra, cranial; ven, ventral; cau, caudal. (Scale bar: 50 μm.)