核酸适配体分子的精准筛选与优化

In recent years, with the rapid development of the research on apatmers and the establishment of advanced screening techniques and screening platforms, significant progress has been achieved both on the speed and efficiency of aptamer screening process. Compared with antibodies, however, the practical application of aptamers still faces significant challenges. How aptamers keep its conformation in real sample systems is still a crucial question in coming years. Considering these difficulties, this project will focuse on four aspects: the design of the molecular libraries, the oriented screening of target molecules, the specificity study of aptamers and the optimization of screened aptamers. Firstly, the diversity of screening libraries is greatly enlarged through utilizing improved libraries including complementary libraries, coupling libraries, nanoprobes-assist libraries and so on; Secondly, aptamers that can recognize multi-sites are obtained according to the oriented screening techniques. The screened aptamers lay the solid foundation for the design of sandwich assays; Thirdly, the specificity study of aptamers explores the influence of complex system on aptamers, thus discovering the interferential molecules. Finally, the optimization of screened aptamers digs up the dominant factor in recognitive process, then provides more theoretical direction in the design of aptamers. The optimized aptamers ensure the improved performance of stability, specific recognition and precise recognition in real sample system. The developed advantages will significantly expand the application of aptamers in real sample system.

近年来，随着核酸适配体研究的迅速发展，特别是多种先进的适配体筛选技术与平台的建立，适配体筛选速度与效率都得到了长足的进步。然而，与抗体相比，适配体的实际应用仍面临着重要的挑战与考验。如何保证适配体在真实体系中的实际应用是目前适配体研究中面临的关键问题。本课题拟围绕适配体筛选中分子文库的设计、靶标分子的定向筛选、适配体特异性以及适配体候选序列优化等内容开展系统研究。通过互补型文库、偶联型文库与纳米探针文库等多种新型文库的建立改善文库的多样性；并基于定向筛选获得多位点识别的适配体，从而为建立适配体夹心式检测提供基础，考察复杂体系对适配体结构的影响，发现影响核酸适配体分子识别的主要因素，为核酸适配体的结构优化提供更多理论指导，实现核酸适配体分子结构在真实体系中的稳定性、适配体识别的特异性以及适配体识别位点的精确性等性能的提高，并有助于核酸适配体在真实体系中的应用与推广。

本课题围绕适配体筛选中分子文库的设计、靶标分子的定向筛选、适配体特异性以及适配体候选序列优化等内容开展了系统研究工作。首先，在研究中对筛选分子库进行了创新设计，研究设计了带有固定茎环结构的新型文库以提高文库序列结构的稳定性；研究设计了十面体纳米银探针用于封闭核酸文库序列尾端固定区域，减少对筛选的干扰。其次，对分子位点识别策略以及精准筛选进行了研究，通过结合蛋白筛选与细菌筛选实现对靶向细菌上特定蛋白酶适配体的精准筛选；通过对靶标的定向固定实现双适配体的精准筛选。另外，对于适配体特异性筛选策略进行了研究，包括利用高特异性界面实现高效筛选；以及通过优化负筛策略提高适配体特异性。最后，对于适配体结构优化新策略进行研究，利用计算机分子模拟技术研究了适配体结构域以及适配体-靶标相互作用，建立了数种适配体应用新方法，可对蛋白、细胞进行高特异性高选择性检测。