基于CE-MS及结合位点导向的核酸适配体Non-PCR直接测序筛选方法研究

Aptamers as novel molecular recognition probes, are one of the research hotspots in biology, food and environment analysis, disease diagnosis and treatment, nucleic acid drug development. Their related research has obvious interdisciplinary characteristics, a wide range of applications and great potential for producing economic benefits. At present, efficient screening of aptamers is a tough nut to crack and bottleneck, and there are no breakthroughs at home and abroad. Based on our ten years of research on aptamer screening mechanism and strategy, now is the time we reassess SELEX procedure. We think that in the screening steps of current SELEX design, multiple rounds PCR results in sequences partial amplification, so a large number of candidate sequences are distorted. Moreover, further plasmids based sequencing results in some sequences missing, and high-throughput sequencing results in the difficulty of mass data analysis. So PCR amplification is the key to affect screening complexity and low success rate. This project proposes a new methodology for aptamers screening, “CE-MS based binding site oriented Non-PCR direct sequencing method for aptamers screening”. The high efficiency separation of “target-nucleic acid” complex by capillary electrophoresis and high resolution mass spectrometry analysis as well as bioinformatics are combined to directly obtain the information of their binding site, binding region, and binding sequence, which abandons the multiple rounds PCR amplification, achieves high fidelity accurate screening of aptamers. The study will be an important revision of SELEX technology and is expected to bring a breakthrough for aptamers screening.

核酸适配体做为新型分子探针，是生物分析、食品和环境检测、疾病诊疗、核酸药物的研究热点之一，具有明显的交叉学科特色和产生经济效益的巨大潜力。核酸适配体的高效筛选是最难啃的骨头和瓶颈，国内外都没有取得明显突破。通过十年的筛选机理和策略研究，我们重新审视SELEX技术，认为筛选步骤中的多次PCR会造成偏性扩增放大和大量候选序列失真，直接导致筛选过程复杂、成功率低，而克隆测序或高通量测序则会引起序列漏选或海量数据的分析。.本项目提出全新的筛选理念和研究思路，建立“基于CE-MS的结合位点导向的核酸适配体Non-PCR直接测序筛选方法”。基于毛细管电泳高效分离“靶标-核酸”复合物，结合高分辨质谱，直接获得复合物中结合靶标位点的适配体区域片段及序列信息。摒弃SELEX中通用的多轮PCR扩增，实现适配体的高保真精准筛选。本研究将是对SELEX技术的重要修正，有望为适配体筛选带来突破。

核酸适配体做为新型分子探针，是生物分析、食品和环境检测、疾病诊疗、核酸药物的研究热点之一，具有明显的交叉学科特色和产生经济效益的巨大潜力。核酸适配体的高效筛选是面临的最大瓶颈，国内外都没有取得明显突破。目前SELEX技术中的多次PCR会造成偏性扩增放大和大量候选序列失真，直接导致筛选过程复杂、成功率低，而克隆测序或高通量测序则会引起序列漏选或海量数据分析困难。. 本项目将高效的毛细管电泳与高分辨结构解析生物质谱联用，借助生物信息学手段，以靶标与核酸的结合位点为导向，完成以结合位点为导向的核酸适配体的高保真准确筛选。建立了基于多模式毛细管电泳（MCE）和高分辨质谱联用的核酸适配体高效筛选平台，毛细管电泳不仅可显著减少筛选轮次和PCR次数，还可以联用质谱仪，对蛋白-核酸复合物进行直接解析；开发了一种无鞘界面的straight nanoESI源，并应用于MCE和Q-TOF质谱仪的耦合。建立了新型无鞘液毛细管电泳-质谱联用接口，适用于核酸库、蛋白质、“蛋白-核酸”复合物的高效分离和高灵敏检测的CE-MS方法。建立了基于天然质谱的结合位点导向的“靶标-核酸”复合物的质谱解析方法，并建立相应的核酸序列分析质谱指纹谱库，为功能蛋白靶标的适配体直接筛选和适配体序列的性能分析提供理论依据。进行了结合位点导向的CE-MS适配体筛选方法与SELEX筛选方法的比较研究，成功探索了结合位点的诱导效应机制，并获得了具有良好RecA ATPase抑制活性的适配体，构建以多元化的竞争体系为基础的适配体鉴定平台，满足更广泛靶标的具有准确生物功能的适配体的筛选需求。