使用单分子荧光显微镜深入研究人体端粒酶的结构和功能

Fully understanding the biochemical action of human telomerase for us is presently a huge challenge since it is a very big complex; moreover, it is remarkably difficult to get enough amount of heloenzyme for traditional enzyme analysis. We must adopt novel methods to assay human telomerase and understand enzymaticaction. Single molecule technique not only can detect dynamic of individual molecule and flexibility of enzyme catalysis, but also can check very little amount of samples in solution or on surface, which cannot be performed by bulk experiment. 1）Single-molecule fluorescence spectroscopy (coincidence spectroscopy, FRET and Pipette delivery to initiate catalysis) will be used for us to investigate the biochemical action of human telomerase. We will adopt step-by-step method to study the complex system. Firstly we may separately investigate hTR and hTERT. Because we can get enough hTR sample for bulk experiment and single molecule detection, we can detailed investigate the structure and function of hTR, especially focus on tertiary and quarterly structure. In studying tertiary structure of hTR, we concern some interesting domains, including pesudoknot domain and Box H/ACA and CR7 domain. 2）we will investigate the interaction between hTR and hTERT. Although scientists believe that human telomerase is complex, at least containing two hTRs. Some specific regions responsible for hTR and hTERT have been identified, however some key questions still remain, such as stoichiometry of hTR and hTERT and active site stoichiometry. These cases involve protein-protein interaction and protein-RNA recognition. Utilizing single-molecule fluorescence coincidence spectroscopy and FRET, we can address some key questions in human telomerase, such as detailed stoichiometry of hTR and hTERT, recognition sequence and the mechanism of protein-RNA interaction and recognition. 3）the most challenging studies in the overall project are to understand the catalytic action of human telomerase. During processive telomere extension, telomerase repeatedly uses the same small integral template for DNA synthesis. There are several key questions we need to answer. At the first stage，telomerase binds its substrate telomere into its active site. How does telomerase adjust its affinity during reaction cycle? The process maybe involves either hTR, or protein, or hTR and protein. The detailed mechanism of recognition and binding between telomere and telomerase also need to be solved. At the second stage, telomerase extend its substrate. We need to investigate the velocity of single round extension in reaction cycle and which factors effects the velocity, such as substrate concentration, counterions concentration and telomere length. At the third stage,The translocation reaction requires a change from a strand-annealing and DNA-synthesizing enzyme to a strand-separating enzyme. This property is different from other polymerases.

人类端粒酶（Human Telomerase）是一种特殊的反转录酶，是用来控制和维护染色体末端单链DNA（Human Telomere）。超过85％的癌细胞中都有活性端粒酶的存在，它是一个典型标记分子，能够作为癌症的治疗靶点。因此，对端粒酶结构和功能的深入研究将是解决细胞老化和细胞永生(癌症)这一世界性生物学难题的基础。人体内端粒酶表达自然丰度低，使用单分子技术才能够帮助建立高分辨率的端粒酶结构模型。本课题提出联合使用独立开发出来的双色激发双色检测单分子显微镜（TCCD）和荧光共振能量转移技术（FRET）深入研究端粒酶，来探讨端粒酶的主要结构构象和分子内局部结构运动，解析出端粒酶的高分辨率结构，和催化功能之间的重要关系，并要在特定时间在特定位置用小分子来启动并控制端粒酶生物催化反应。目标是找出这种酶与人体肿瘤产生的直接或间接的因果关系，从而提供能治疗人类恶性疾病的可行路径。

本研究主要采用双色激发双色检测单分子显微镜（TCCD）和荧光共振能量转移技术（FRET）深入研究了自然表达丰度低，缺失高分辨率结构的端粒酶。首次实现：. （1）构建出高特异性响应癌细胞内端粒DNA分子的传感器，完成了端粒和端粒酶的表达和标记、酶化合物表征等工作，成功进行了TCCD-FRET技术应用于进行端粒酶化合物表征；. （2）使用荧光技术检测跟踪in vitro端粒酶并探索端粒酶活性分子机理，完成单分子技术对生物催化反应的有效控制等工作；. （3）完成TCCD-FRET与定时定位技术的合并并使用它来研究端粒酶的主要结构构象和分子内局部结构运动等工作；. （4）探讨了端粒酶检测创新技术与环境中污染物可能致癌的科学问题之间的关系。该研究属于高度交叉学科项目，我们通过对石墨烯、碳纳米管、重金属等材料进行修饰及改性，成功制备了具有强吸附性能的石墨烯、碳纳米管等新型碳基复合材料，实现了水体中抗生素等有机污染物的高效去除及高灵敏检测，同时构建了检测细胞中的生物小分子的高灵敏、高选择性的纳米传感器。. 本研究部分揭示出端粒酶和环境污染与人体肿瘤产生的直接或间接的因果关系，从而提供了治疗人类恶性疾病的可行路径。