Laboratory of RNA Chemical Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
Anal Biochem. 2011 Oct 1;417(1):112-21. doi: 10.1016/j.ab.2011.05.037. Epub 2011 May 30.
The labeling of newly synthesized DNA in cells to identify cell proliferation is an important experimental technique. The most accurate methods incorporate [(3)H]thymidine or 5-bromo-2'-deoxyruidine (BrdU) into dividing cells during S phase, which is subsequently detected by autoradiography or immunohistochemistry, directly measuring the newly synthesized DNA. Recently, a novel method was developed to detect DNA synthesis in proliferating cells based on a novel thymidine analog, 5-ethynyl-2'-deoxyuridine (EdU). EdU is incorporated into DNA and subsequently detected with a fluorescent azide via "click" chemistry. This novel technique is highly sensitive and does not require DNA denaturation. However, it was also found that EdU exhibits time-dependent inhibition effects on cell growth. Therefore, here we report a novel deoxycytidine analog, 5-ethynyl-2'-deoxycytidine (EdC), that can be used to detect DNA synthesis in vitro and in vivo at a similar sensitivity level compared with EdU. Furthermore, the EdC-induced cytotoxicity is much less than that of EdU when combined with thymidine. This will be a potential application for the long-term detection of proliferating cells.
将新合成的 DNA 标记在细胞中以识别细胞增殖是一项重要的实验技术。最准确的方法是在 S 期将 [(3)H]胸苷或 5-溴-2'-脱氧尿苷 (BrdU) 掺入分裂细胞中，然后通过放射自显影或免疫组织化学直接检测新合成的 DNA。最近，开发了一种新方法来检测增殖细胞中的 DNA 合成，该方法基于一种新型胸苷类似物，5-乙炔基-2'-脱氧尿苷 (EdU)。EdU 掺入 DNA 中，随后通过“点击”化学与荧光叠氮化物结合进行检测。这种新技术非常灵敏，不需要 DNA 变性。然而，也发现 EdU 对细胞生长具有时间依赖性抑制作用。因此，我们在这里报告了一种新型的脱氧胞苷类似物，5-乙炔基-2'-脱氧胞苷 (EdC)，它可以在体外和体内以与 EdU 相似的灵敏度水平检测 DNA 合成。此外，当与胸苷结合时，EdC 诱导的细胞毒性比 EdU 小得多。这将是长期检测增殖细胞的潜在应用。
