适配体靶向与转运的功能化核酸探针构建及端粒酶活性调控与分析

Numerous studies have shown that human telomerase activity is closely related to tumor development. Therefore, telomerase can be regarded as a potential target for anticancer drug design and molecular markers for early diagnosis of cancer. Conventional nucleic acid probe-based functional regulation of telomerase facing two challenges: 1) nucleic acid probes are susceptible to nuclease degradation; 2) inefficient cellular uptake of nucleic acid probes. Based on the high express of nucleolin on tumor cell membrane and the recent progress on nucleolin aptamer mediated cell uptake and dilivery, this project proposed new methods and technologies for the regulation of telomerase biological function and activity assay. Regulation of telomerse can be achieved with high nuclease tolerance and high delivery efficiency by the construction of thio-phosphoramidate modified nucleolin aptamer-telomerase RNA antisense oligonucleotide probe. Through the study and optimization of some key indicators, such as biological compatibility of the nucleic acids probes, efficiency of celluar uptake, subcellular localization，stability of nucleic acids probes, and intracellular regulation by confocal fluorescence techniques, fluorescence auto/ cross-correlation spectroscopy, etc., the biological effects and the treatment and diagnosis potential of the tumor with telomerase as the target can be evaluated. With further design of isothermal amplification and paper based telomerase activity assay, it is eventually hope to establish a novel technological system integrating a series of processes from telomerase activity regulation to telomerase activity analysis. The goal of this study is expected to provide a new foundation and perspective for clinical medicine, pharmacy, analytical chemistry, and other fields.

大量研究表明人端粒酶活性与肿瘤发生发展密切相关，所以端粒酶可视为一种潜在的抗肿瘤药物设计靶点以及癌症早期诊断分子标志物。当前基于核酸探针的端粒酶功能调控研究面临两个难题：1）核酸探针易受细胞内核酸酶的降解；2）核酸探针低效的细胞传输。本项目基于核仁素在癌细胞膜高表达特性及适配体细胞转运的新进展，拟构建高核酸酶耐受性、高效细胞传输特性的巯代修饰适配体-端粒酶RNA反义核酸探针，用于端粒酶活性调控。以激光共聚焦、荧光自/互相关光谱等技术手段评价并优化探针的生物兼容性、摄入效率与细胞器定位、细胞内稳定性、调控时效等关键性指标与参数，并评价以端粒酶为靶点的肿瘤细胞的生物学效应、以及肿瘤治疗、检测新潜能。通过进一步发展等温扩增技术的端粒酶活性试纸条检测新方法，希望最终实现从活性调控与检测于一体的端粒酶生物学研究新技术。本研究有望为临床医学、药学、分析化学等领域研究端粒酶生物学提供新的研究基础与视角。

自2014年获得本项目资助以来，本项目研究团队围绕 “核酸与蛋白分析”这一主线，系统开展包括端粒酶活性分析在内的高灵敏生物医学分析与传感新原理新方法研究。在涉及到端粒酶、疾病诊断、病原微生物分析方法的背景下，在一些关键技术和方法上取得了若干进展，具体表现为电化学发光平台和探针构建、发展纸基基因检测芯片及生物分析应用、核酸扩增（信号扩增）新方法等，在本项目资助下共发表了16篇论文，大部分成果发表在本领域的核心刊物，如Analytical Chemistry, Biosensors and Bioelectronics, Lab on a Chip等上。有些技术申请了国家发明专利，其中已经授权了1项。本项目发展的纸基因检测芯片在食品安全检测、疾病诊断领域具有产业化潜力。相关工作也得到了学术界广泛的认可，国内外学术界多次引用并高度评价本项目研究工作。在包括国际电化学发光大会（法国波尔多），国际生物发光和化学发光大会（法国南特），中国化学会年会，分析化学年会等上多次展示本项目成果。