构象调控的组合核酸适体多靶标筛选新方法研究

Aptamers that are selected in vitro from random DNA or RNA libraries by SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique have been extensively explored recently because of their potential applications in the areas such as analysis and biomedicine. Although research of aptamers has made great progress during the past decades, the aptamer selection is still the main bottleneck of this area. In order to separate bound sequences from unbound sequences during the selection procedure, targets are usually immobilized on solid support. The immobilization always changes the structure and properties of targets and results in the lower affinity and specificity of the obtained aptamers. The sensitivity and specificity of aptamer probes are often compromised because of the introduction of an allosteric element. In this proposal, we aim to develop a novel selection method for allosteric aptamers by combining a streptavidin binding aptamer as an allosteric element. By this method, allosteric combined aptamers against non-fixed multi-targets can be generated simultaneously. The obtained allosteric aptamers can be used directly in detection and separation of targets without post-optimization or construction. The conventional aptamers can easily be obtained through removing the streptavidin binding aptamer from the combined aptamers. This study includes the design of DNA libraries, optimization of selection process, characterization of aptamer candidates and applications of combined aptamer in the real samples. We also investigate the foundation of aptamer selection, such as the laws of aptamer evolution during selection process and the structure-affinity relationship of aptamers.

核酸适体是从随机核酸文库中筛选得到的可与靶标特异性结合的单链寡核苷酸，在分离、分析以及生物医学领域有着广泛的应用前景。虽然核酸适体研究已取得较大进展，但其筛选仍然是该领域的瓶颈。筛选过程中往往要将靶分子固定，固定后靶分子的结构和性质会发生变化，导致得到的核酸适体与游离靶分子的亲和力与特异性降低。核酸适体探针的构建往往引入构象调控单元，也会导致其亲和力与特异性降低，影响实际应用。针对筛选和应用中的问题，本项目拟以我们筛选的链霉亲和素核酸适体为构象调控元件，建立构象调控的组合核酸适体筛选新方法，以实现免固定、多靶标同时筛选；所得到的组合核酸适体无需进一步设计，可直接用于靶标的分离与检测；去掉其构象调控部分即可获得常规的核酸适体。研究工作包括新型DNA文库的设计、筛选过程的优化、核酸适体的表征及其分析应用；同时对核酸适体筛选的进化规律，及其与靶分子相互作用的构效关系等基本问题展开基础研究。

核酸适配体是从随机核酸文库中筛选得到的可与靶标特异性结合的单链寡核苷酸，在分离、分析以及生物医学领域有着广泛的应用前景。本课题针对核酸适配体在筛选和应用方面的瓶颈问题，开展了活细胞等复杂靶标的筛选、核酸适配体的表征与优化、融合调控核酸适配体的设计及核酸适配体靶标的鉴定等相关应用基础研究。筛选得到了特异性识别前列腺癌的核酸适配体Wy-5a，其对靶细胞的平衡解离常数为74 nM，与恶性前列腺癌组织切片有高的特异性结合，而与其他多数肿瘤细胞系不结合，该核酸适配体可望用于前列腺癌的诊断、预后或靶向治疗；筛选得到了耐药乳腺癌MCF-7R细胞的适配体M17A2，其对靶细胞的平衡解离常数达到2.06±0.28 nM，其结合于靶细胞之间的连接处和细胞间隧道纳米管上的某种蛋白，可能与细胞通讯有关。我们构建了ATP调控的链霉亲和素融合构象调控核酸适配体，实现了对50-1000μM 浓度范围内的ATP 的免标记快速分离与检测，这种策略可用于构建其他的构象调控核酸适配体探针并有望应用于实际应用。基于核酸适配体的肿瘤生物标志物的发现是肿瘤研究的一个前沿领域。识别细胞的核酸适配体的靶标鉴别是核酸适配体生物标志物发现研究的瓶颈问题，我们建立了一个基于SILAC定量蛋白质组学的可监控的核酸适配体靶蛋白鉴别的通用性的新方法，鉴定出核酸适配体Sgc-3b和Sgc-4e的靶标分别为L-选择素和整合素a4，这两个靶标都是白血病的潜在生物标志物。这个通用的核酸适配体靶标的鉴定方法可以促进核酸适配体的靶标鉴定的研究，进而促进核酸适配体的实际应用。