基于核酸酶反应的生物模拟核酸适配体筛选技术研究

项目名称： 基于核酸酶反应的生物模拟核酸适配体筛选技术研究

项目编号： No.21305093

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 数理科学和化学

项目作者： 娄新徽

作者单位： 首都师范大学

项目金额： 25万元

中文摘要： 核酸适配体(aptamer)在生物传感和生物医学领域具有重要的应用价值。缺乏快速高效的筛选技术是制约其广泛应用的最主要原因之一。我们近期的研究发现aptamer与靶标的复合物可以很好地抵制多种核酸酶降解（AC，2012）。同时基于我们在适配体筛选技术方面的经验和发现的问题（PNAS，2009，2010），本项目提出一种基于核酸酶反应的aptamer筛选新方法（Enzymatic -SELEX）。其核心创新点是模拟生物体系，利用核酸酶的结构选择性将不能与靶标结合的单链DNA链降解，只有与靶标结合的aptamer进入下一循环的筛选。该技术无需任何分离设备，且无需将靶标或文库进行固相固定，操作简单廉价。此外采用无引物文库，消除引物区对筛选影响，有利于大幅提后筛选过程中aptamer结合区域分析的效率。本项目将对发展我国具有自主产权的适配体筛选技术、生物传感技术及疾病诊断与治疗技术有着重要意义。

中文关键词： 核酸适配体；筛选技术；邻苯二甲酸酯类增塑剂；生物传感器；界面效应

英文摘要： Aptamers hold great promise in biosensing and biomedical fields. One of the major obstacles that prevent the wide applications of aptamers is the lack of rapid and highly efficient aptamer selection approaches. We recently reported that the aptamer-target complex can resist the digestion of various nucleases. Based on our previous work on developing aptamer selection methods (PNAS, 2009, 2010), we proposed here a nuclease reaction-based method for the selection of aptamers, called Enzymatic-SELEX. The core novelty of the proposed method is that by mimicking biological system, the unbounded DNA sequences are digested by nuclease and only the oligonucleotides that bound with the target molecules are survived for the next cycle of selection. This method, simple for operation and low-cost, doesn't require any instruments for separation and doesn't need to immobilize target onto the solid phase as well. In addition, the use of primer-free DNA pool in the selection can eliminate the interference of PCR primer regions on target binding, improve post selection efficiency in identifying aptamer-target binding regions. The method proposed here would make important contribute to the development of aptamer selection technology, sensing technology, and disease diagnostic and treatment technologies.

英文关键词： Aptamer；SELEX；Phthalic acid esters；Biosensor；Surface effect