Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides
Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides
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DOI：
10.1073/pnas.1101126108
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发表时间：
2011-12-20
影响因子：
11.1
通讯作者：
Luedtke, Nathan W.
中科院分区：
文献类型：
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作者：
Neef, Anne B.;Luedtke, Nathan W.
常用的DNA代谢标记物，包括5-乙炔基-2‘-脱氧尿苷(EDU)和BrdU，都是有毒的抗代谢药物，会导致DNA不稳定、坏死和细胞周期停滞。除了扰乱生物功能外，这些特性还可以阻止需要后续组织存活的代谢标记研究。为了绕过导致毒性的代谢途径，同时保持被代谢结合到DNA中的能力，我们合成并评价了一个小家族的阿拉伯呋喃葡萄糖-乙炔基尿嘧啶衍生物。其中，(2‘-S)-2’-脱氧-2‘-氟-5-乙炔基尿苷(F-Ara-EDU)在不同组织类型中具有选择性标记作用，但对基因组功能影响最小。利用铜(I)催化的叠氮化物-乙炔与荧光叠氮化物的“点击”反应，可以很容易地检测到F-Ara-EDU与DNA的代谢结合。F-Ara-EDU的毒性比BrdU和EDU都低，在需要长期细胞存活和/或深层组织成像的实验中可以更灵敏地检测到它。与以前报道的2‘-阿拉伯修饰核苷和EDU不同，F-Ara-Edu很少或根本没有引起细胞停滞或DNA合成抑制。因此，F-Ara-Edu非常适合于以活体DNA为目标的脉冲追逐实验。作为示范，斑马鱼胚胎在单细胞期显微注射F-Ara-EDU，并在受精后10h被BrdU追赶。发育3d后，在脊索背侧和上皮细胞中观察到仅含F-Ara-EDU的静止期/衰老期细胞的复杂形态。因此，阿拉伯糖基核苷衍生物为将生物正交官能团引入DNA提供了独特而有效的手段，在基础研究、生物技术和药物开发中有着广泛的应用。
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.