基于多模式毛细管电泳的核酸适配体三维度筛选策略研究

Aptamers can be used as recognition probes in biological, food and environment analysis. Moreover, they have great potential in disease diagnosis and treatment, drug discovery, medicine research, as well as bioimaging, which are expected to bring huge economic benefits. However, current aptamers are far from satisfactory, and have not yet been fully developed. The complicated selection process with high cost and low efficiency is one of the bottlenecks of their application. There is still not universally accepted standard selection methods for proteins, cells, microorganisms and small molecules targets..In this project, we aim at the strategy research of aptamer selection against multiple targets based on capillary electrophoresis (CE), which has the characteristics of high efficiency, high speed, low cost and multiple modes. With high throughput sequencing and bioinformatics methods, the sequence compositions, structure characteristics and biochemical properties of aptamers will be studied, and the availability of randomly synthesized nucleic acid libraries will be evaluated; we hope to establish several interaction models of "target-library" as well as parameters and impacting factors for quantitative evaluation of the "target-library" binding; the strategy research of aptamer selection will be done from three dimensions: “the integration of selections based on affinity and specificity”, “the optimization and design of selection conditions”, “the application of multiple modes of CE”; we intend to improve the multiple-mode CE platform and establish a CE fingerprint library for aptamers selection. We hope above progresses can provide efficient and useful aptamer selection strategy as well as universal rule for different scales targets of proteins, cells, microorganisms and small molecules.

核酸适配体可作为生物分析、食品和环境检测的探针，并在疾病诊断和治疗、药物研发和分子成像领域具有广阔的应用空间和产生经济效益的巨大潜力。目前的适配体远未发挥潜能。筛选复杂，成本高，效率低是其应用受限的瓶颈之一。针对蛋白质、细胞、微生物和小分子等多种靶标，尚无普遍接受的标准筛选方法。.本项目基于高效、快速、低成本、模式多的毛细管电泳技术进行适配体筛选策略研究。结合高通量测序和生物信息学方法研究适配体的序列组成、结构特征和生化性质，进行随机核酸库的有效利用率评价研究；建立“靶-库”相互作用模型，“靶-库”结合的定量评价指标和影响因子等参数；从“亲和力和特异性筛选的组合”、“筛选条件优化和设计”及“多模式毛细管电泳筛选方法应用”三个维度进行筛选策略研究；建立和完善多模式毛细管电泳筛选平台和指纹图谱库。为蛋白质、细胞、微生物、小分子多尺度靶标的核酸适配体筛选提供具有一定普适性的有效实用的筛选策略。

本课题建立了三种创新的毛细管电泳筛选模式：包括一步在线反应CE筛选、同步竞争CE筛选、在线竞争荧光恢复筛选等，解决了同步正负筛选、多靶筛选、小分子筛选等难题。评价了40种靶蛋白与初始核酸库结合的电泳图谱，建立了“靶-库”相互作用模型、“靶-库”相互作用的表征方法和定量评价参数体系，提出了不同靶蛋白的分类评价方法和相应的筛选策略。已筛选获得18种重要功能蛋白质的核酸适配体，其中12种蛋白的核酸适配体是首次报道。并建立了多种基于纳米材料的核酸适配体应用方法。特别是，在2020年的新冠疫情爆发期间，两周内快速筛选了新冠病毒刺突蛋白Spike及其受体ACE2的适配体。通过本课题的研究，所建立的核酸适配体筛选策略和方法具有创新性、普适性及应用价值，使核酸适配体的筛选时间和资金成本显著降低。