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PNAS:科学家发明体内DNA合成可视化新技术

2011/12/15 来源:科学网
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瑞士苏黎士大学的研究人员研发了一种新物质,可用来标记和观察动物体内的DNA合成过程。该技术的应用为药物研发提供了新策略。相关研究论文于12月5日在线发表在美国《国家科学院院刊》(PNAS)上。

瑞士苏黎士大学的研究人员研发了一种新物质,可用来标记和观察动物体内的DNA合成过程。该技术的应用为药物研发提供了新策略。相关研究论文于12月5日在线发表在美国《国家科学院院刊》(PNAS)上。

详细了解动物体内DNA和蛋白质等大分子合成是理解生物系统和设计疾病治疗策略的必要条件。通常,通过人工合成小分子标记物掺入生物体自身合成过程来达到可视化DNA合成的目的。但是,直到现在该方法有一个重大的局限性:标记物具有毒性并导致细胞死亡。内森·利德基(Nathan Luedtke)领导的小组研发了一种叫“F-ara-Edu”的核苷。用它来替换胸腺嘧啶脱氧核苷,标记DNA对生物体基因组功能几乎没有影响,毒性也大为降低,检测也更灵敏。

利德基表示,通过可视化新DNA的合成,就能够鉴定病毒感染和肿瘤增长的位点。这将引领药物研发新策略。


Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides

Neef, Anne B.; Luedtke, Nathan W.

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.

文献链接http://www.pnas.org/content/early/2011/11/28/1101126108

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