Telomerase is a ribonucleoprotein complex that is usually activated in the cancer cells and is closely related to telomere maintenance and immortalization of cancer cells. Telomerase activity detection is important for early diagnosis of human cancers as well as the screening of telomerase-target anti-cancer drugs. A new simple and fast method to detect the telomerase activity has been developed based on hybridization chain reaction (HCR) and magnetic separation. In the assay of experiment, the biotin-labeled telomerase substrate is elongated by telomerase generating a special DNA with repeated sequences-(ggttag)n at their terminals. These telomerase elongated products are fixedly connected with streptavidin-coated magnetic beads through the specific combination of streptavidin with biotin. At the same time, other cell extracts are removed by magnetic separation. A specific DNA probe I is designed as the initiator of HCR. 3′-Terminus of DNA probe I is complementary with three repeated sequences of telomerase elongated product. So, DNA probe I could be fixed on magnetic bead through hybridization. 5′-Terminus of DNA probe I is in charge of triggering HCR with DNA probe II and probe III. DNA probe II and probe III are modified with fluorophores. So, the HCR amplification results can be easily detected by fluorescence. All of excessive DNA probes can be removed by magnetic separation. Under the optimal conditions, telomerase activity in 1.0×10 Hela cells has been obviously detected. Because no enzyme involves in the signal amplification process of HCR, our proposed method can effectively avoid the interference of nonspecific amplification which usually exists in the polymerase amplification processes and increase the accuracy of the test results. Furthermore, enzyme-free signal amplification can effectively avoid the potential interference of telomerase inhibitor to the enzyme activity and improve the reliability of screening of telomerase inhibitors.

中文翻译：

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基于杂交链反应信号放大结合磁分离的端粒酶活性检测

端粒酶是一种核糖核蛋白复合物，通常在癌细胞中被激活，与癌细胞的端粒维持和永生化密切相关。端粒酶活性检测对于人类癌症的早期诊断以及端粒酶靶向抗癌药物的筛选具有重要意义。基于杂交链反应 (HCR) 和磁分离，开发了一种新的简单快速检测端粒酶活性的方法。在实验分析中，生物素标记的端粒酶底物被端粒酶延长，产生特殊的DNA，末端有重复序列-(ggttag)n。这些端粒酶延长产物通过链霉亲和素与生物素的特异性结合，与链霉亲和素包被的磁珠固定连接。同时，其他细胞提取物通过磁分离去除。特异的 DNA 探针 I 被设计为 HCR 的启动子。DNA 探针 I 的 3'-末端与端粒酶延长产物的三个重复序列互补。因此，DNA探针I可以通过杂交固定在磁珠上。DNA 探针 I 的 5'-末端负责用 DNA 探针 II 和探针 III 触发 HCR。DNA 探针 II 和探针 III 用荧光团修饰。因此，可以很容易地通过荧光检测 HCR 扩增结果。所有过量的 DNA 探针都可以通过磁分离去除。在最佳条件下，在1.0×10 Hela细胞中可明显检测到端粒酶活性。由于没有酶参与 HCR 的信号放大过程，我们提出的方法可以有效避免聚合酶扩增过程中通常存在的非特异性扩增的干扰，提高检测结果的准确性。此外，无酶信号放大可以有效避免端粒酶抑制剂对酶活性的潜在干扰，提高端粒酶抑制剂筛选的可靠性。