Quantitative Determination of Telomerase Activity by Combining Fluorescence Correlation Spectroscopy with Telomerase Repeat Amplification Protocol.
Quantitative Determination of Telomerase Activity by Combining Fluorescence Correlation Spectroscopy with Telomerase Repeat Amplification Protocol.
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DOI：
10.1021/acs.analchem.7b04256
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发表时间：
2018-01
影响因子：
7.4
通讯作者：
Di Su;Xiangyi Huang;Chaoqing Dong;Jicun Ren
中科院分区：
文献类型：
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作者：
Di Su;Xiangyi Huang;Chaoqing Dong;Jicun Ren
端粒酶是维持端粒长度的关键酶，因其在癌症和衰老中的重要作用而被认为是一种多功能的癌症生物标志物和潜在的药物靶点。由于端粒酶活性在细胞中的水平很低，因此有必要建立一种灵敏可靠的检测方法。在这篇文章中，我们提出了一种结合单分子荧光相关光谱(FCS)和端粒酶重复序列扩增法(TRAP)的超灵敏和可靠的端粒酶活性定量检测方法。这种新方法(FCS-TRAP)的原理是基于FCS测量TRAP产物的特征扩散时间和分子数的变化。特征扩散时间与陷阱产物的长度有关，分子数代表陷阱产物的浓度。我们优化了捕集程序和FCS测量的条件。我们观察到，在最佳条件下，端粒酶活性与特征扩散时间和TRAP产物的分子数呈正相关。该方法成功地用于不同细胞端粒酶活性的检测，实现了单个细胞的检测。同时，用该方法对缓蚀剂的缓蚀效果进行了评价，得到的IC50值与文献值吻合较好。与现有的捕集方法相比，该方法具有定量可靠、灵敏度高、检测时间短、不需要分离等优点。我们认为，FCS-TRAP方法在端粒酶抑制剂的临床诊断和筛选中具有潜在的应用价值。
Telomerase is a key enzyme for maintaining the telomere length and is regarded as a versatile cancer biomarker and a potential drug target due to its important role in cancer and aging. It is necessary to develop a sensitive and reliable method for detection of telomerase activity due to its very low level in cells. In this Article, we propose an ultrasensitive and robust method for quantitative determination of telomerase activity by combining single molecule fluorescence correlation spectroscopy (FCS) with telomerase repeat amplification protocol (TRAP). The principle of this new method (FCS-TRAP) is based on measurement of the change in characteristic diffusion time and molecule number of TRAP products by FCS. The characteristic diffusion time is related to the length of TRAP products, and the molecule number represents the concentration of TRAP products. We optimized the conditions of TRAP procedure and FCS measurements. We observed that the telomerase activities are positively correlated to characteristic diffusion time and molecule number of TRAP products at optimal conditions. This method was successfully used for determination of telomerase activity of different cells, and detection of a single cell was realized. Meanwhile, this method was used to evaluate the inhibition efficiency of inhibitors, and the IC50 values obtained were in good agreement with the references. Compared to current TRAP methods, this method shows reliable quantification, ultrahigh sensitivity, and short detection time and is without separation. We believe that the FCS-TRAP method has a potential application in clinical diagnosis and screening of telomerase inhibitors.