纳米孔单分子技术应用于端粒酶活性检测的研究

Telomere length and telomerase activity are closely associated with cell aging and carcinogenesis. It has been found that there was overexpression of telomerase in 85% of known tumors, and thus telomerase is regarded as one of the biomarkers for cancer early diagnosis and targeted therapy. Therefore, it is highly desirable to establish a simple and sensitive method for measuring the activity of telomerase which is vital for cancer early diagnosis and therapy. In this project, we aim to use the characteristic current events generated during nanopore sensing for measuring the telomerase activity. A DNA strand elongated by telomerase forms a duplex structure with a DNA probe. Translocation of the duplex through -hemolysin nanopore would generate highly characteristic current events. Statistical analyses of the current frequency and dwell time will reveal the information of the telomerase activity and also the length of the repeated elongation. This method does not rely on PCR or fluorescent labeling, and features no background interference and low false negative responses. It is a rapid, sensitive and highly specific approach for measuring telomerase activity suitable for screening of massive samples and high-throughput detection. It may play an important role in telomere studies and the related drug screenings.

端粒长度和端粒酶活性的变化与细胞衰老和癌变密切相关。研究结果发现在已知的85％的肿瘤当中都发现了端粒酶的高表达，端粒酶也被认为是癌症早期诊断和靶向治疗的生物标志物。因此建立灵敏度高、操作简单的端粒酶活性检测方法，对于癌症的早期诊断及治疗具有重要意义。本项目首次基于纳米孔技术用特征信号的方法定量检测端粒酶的活性。在端粒酶的作用下，扩增后的DNA引物可与DNA探针形成双链，穿越-溶血毒素纳米孔时产生特征电流信号，通过特征电流信号的频率和时间参数的变化，不仅可获得端粒酶活性的信息，也可判断扩增重复序列的长度。该方法不用PCR和荧光标记，无背景干扰，假阴性率低，简单快速，灵敏度高、特异性强并能够用于大量样品的快速筛选和高通量平行检测，是对现有方法的一个强有力的补充，对端粒酶活性和端粒的研究及其相关药物的筛选具有重要的意义。

端粒长度和端粒酶活性的变化与细胞衰老和癌变密切相关。研究结果发现在已知的85％的肿瘤当中都发现了端粒酶的高表达，端粒酶也被认为是癌症早期诊断和靶向治疗的生物标志物。因此建立灵敏度高、操作简单的端粒酶活性检测方法，对于癌症的早期诊断及治疗具有重要意义。本项目首次基于纳米孔技术用特征信号的方法定量检测端粒酶的活性。在端粒酶的作用下，延长后的DNA引物穿越-溶血毒素纳米孔时产生特征电流信号，通过特征电流信号的频率和时间参数的变化，不仅可获得端粒酶活性的信息，也可判断扩增重复序列的长度。该方法不用PCR和荧光标记，无背景干扰，假阴性率低，简单快速，灵敏度高、特异性强并能够用于大量样品的快速筛选和高通量平行检测，是对现有方法的一个强有力的补充，对端粒酶活性和端粒的研究及其相关药物的筛选具有重要的意义。另外，本项目还基于纳米孔技术发展了其它酶的检测方法，包括: (1) 建立针对氨肽酶和pH同时检测的纳米孔单分子检测方法; (2) 三链DNA分子信标用于VEGF和MMP9的同时检测; (3) 纳米孔技术应用于多酶体系中底物通道效应的研究。