基于构型固定化的核酸适配体优化及石房蛤毒素的高灵敏度SPR传感检测研究

Aptamer is a kind of single-stranded nucleic acid recognition element, which is selected in vitro. It has broad application prospect in the field of food safety detection, especially for small molecular targets. However, the present selected aptamers of small molecular targets have limited affinity. We analyze the thermodynamical reason herein. It is hard for the highly flexible aptamer with variable configuration to fold into stable conformational structure, and there is large entropy loss when it binds with the target, and thus leading to weak binding ability (affinity). Based on the analysis, we innovatively propose to fix the configuration of aptamer so as to reduce the entropy loss in the binding process, and thus to improve the affinity. In the other words, we will explore to get a fixed configuration by regulating the base distance and sequence in the terminals of the aptamer, and adding multivalent cations. There will be three research contents in the project. Firstly, we will develop a DNA tetrahedron-based SPR assay which can be used for accurate evaluation of the affinity of aptamer; Secondly, we will choose the present aptamer of saxitoxin, APTSTX, as a model, and study the influence rule of the sequence and the multivalent cations on affinity; Finally, the optimized aptamer with highest affinity will be used to develop a SPR-aptasensor for highly sensitive detection of saxitoxin in real food. This project is of great significance to promote the theoretical research and application progress of aptamer.

核酸适配体是经体外筛选获得的单链识别因子，在食品安全领域具有广阔的应用前景，对小分子靶标的检测优势突出。但目前筛选到的小分子的适配体大多亲和力有限，不易实现高灵敏检测。本项目从热力学角度进行分析，高灵活性的适配体链构型多变，难以形成稳定的折叠结构，结合靶标时熵损失严重，故结合能力弱。基于此，本项目提出通过优化使适配体构型固定以减少熵损失，从而提高其亲和力的新思路。即，通过调节适配体末端的距离和序列、调节多价阳离子条件，得到最适固定构型，减少结合熵损失，从而提高亲和力。研究内容包括，采用DNA四面体修饰的SPR芯片建立精准评价适配体亲和力的关键方法；以石房蛤毒素的适配体APTSTX为起始链，进行适配体构型优化，并探讨末端序列、阳离子条件影响亲和力的规律；以亲和力最高的优化适配体构建适配体-表面等离子共振传感器进行石房蛤毒素的高灵敏度检测。本项目对推进适配体的理论研究和应用开发具有重要意义。

核酸适配体具有体外SELEX筛选、成本低、可接受多种化学修饰等优点，在食品分析领域应用前景广阔，但目前筛选的核酸适配体多数亲和力有限，用于构建核酸适配体传感器时，难以满足高灵敏度检测分析。探究制备高亲和力核酸适配体的有效方法，在明确核酸适配体分子识别机制前提下理性设计核酸适配体传感器，是推动核酸适配体走向应用的首要途径。. 本项目从核酸适配体与靶标的结合机理切入，设计了锁定适配体双末端、双适配体嵌合、先截短后双适配体嵌合等post-SELEX策略优化核酸适配体构型、促进核酸适配体的三维折叠，以提高核酸适配体的亲和力，并利用分子对接和分子相互作用研究深入解析所得核酸适配体与靶标的识别机理，最后将高亲和力核酸适配体与高灵敏响应传感技术相结合，开发可针对实际食品样品检测的核酸适配体传感器。通过上述post-SELEX策略，可使得核酸适配体亲和力显著提高，亲和力常数Kd由µM水平降至低nM水平，获得了针对石房蛤毒素等食品靶标的多条高亲和力核酸适配体。在明确了其分子识别机制之后，通过结合纳米四面体辅助所得核酸适配体的理性固定、基于分子识别机制设计理性竞争以及构建高灵敏信号放大策略，成功构建了一系列检测限低至低nM或pM水平的核酸适配体传感器。所构建的核酸适配体传感器具有良好的准确性、选择性，且成本低、操作简便，可用于针对实际食品样品的高灵敏传感分析检测。本项目对于推广核酸适配体的发展和实际应用具有重要的学术意义。