基于三维结构的链霉亲和素适配体的设计优化及用于生物样品中奥沙利铂测定方法的研究

Streptavidin aptamer has continuously generated lots of interest in bio-analytical field, such as detection of RNA, RNA binding proteins, heavy metal ions and small molecules. SELEX method is time-consuming and high-cost for aptamer optimization, and it often fails to achieve the expected aims. In the previous research, a breaking through of crystallization of streptavidin-aptamer (SA-Apt) binary complexes had been gained. Three-dimensional structure based aptamer design and optimization is proposed based on the above research progress. A comprehensive study of SA-Apt binding mode will be executed through X-ray structure elucidation of SA-Apt complexes in this project. Furthermore, platinum (Pt) based anti-tumor drug is selected as a model molecule for 3D-structure based aptamer optimization. SA-Apt binary complex structural model can be used for the preliminary optimization the sequence of platinum detection aptamer probe. Crystallization and X-ray 3D-structure elucidation of SA-probe-Pt ternary complex will be carried out, and further optimize the sequence of the probe based on the ternary complex structure. The sensitivity of the aptameric platform for platinum detection can be improved by increasing the probe affinity to SA and Pt utilization efficiency. A high sensitive platinum detection method will be constructed and used to determine the concentration of oxaliplatin in biological samples. We aim to provide both a general structural model for various streptavidin aptamer optimization and a general technology for aptamer based pharmaceutical analysis.

链霉亲和素适配体广泛应用于RNA及其结合蛋白、重金属离子、小分子药物等分析。应用传统的SELEX方法优化适配体具有盲目性，耗费大、效率低，且往往达不到预期目的。本课题在链霉亲和素-适配体（SA-Apt）二元复合物结晶取得突破性进展的基础上，拟通过解析复合物晶体三维结构，阐明影响两者结合模式的关键因素，提出基于三维结构理性高效设计优化适配体的方法；以铂类药物为研究模型分子，利用SA-Apt复合物结构初步优化铂检测适配体探针序列，结晶并解析链霉亲和素-探针-铂（SA-Probe-Pt）三元复合物结构，精细优化探针序列，提高探针亲和力和对铂的利用效率，最终建立高灵敏的分析方法，用于生物样品中奥沙利铂的测定。本研究将为不同分析方法和应用中链霉亲和素适配体的设计优化提供通用结构模型，且从分子水平阐明了SA-Probe-Pt三元复合物的作用机制，为基于适配体的药物检测方法提供通用技术。

链霉亲和素适配体广泛应用于生命分析领域，链霉亲和素与适配体（SA-Apt）复合物三维结构的解析将为其进一步理性优化设计提供结构模型，本项目在前期研究的基础上，对SA-Apt复合物的结晶进行了系统的筛选和优化，使该复合物晶体的弱衍射范围从120度缩小到50度，但仍无法完整解析其结构，仅明确链霉亲和素与适配体的结合比例为1:1；MicroRNA（miRNA）与多种疾病的发生、发展密切相关，是重要的疾病诊断和治疗的生物标志物，我们开发了一种以链霉亲合素适配体分子信标构象转化为基础，等温链替换反应为放大平台的简单、快速、灵敏度高、成本低的miRNA检测方法，解决了目前miRNA检测定量精度不高、操作复杂、仪器要求高等问题；另外，我们在基于链霉亲和素适配体构象转化检测铂离子的基础上，引入化学发光能量转移技术，构建了一种新颖简便、且特异性和灵敏度更高的铂离子检测方法，有望应用于医学和环境铂离子化合物的检测，相关的适配体序列的特定设计，也为该技术拓展至其他金属离子或蛋白质的检测提供了一种方法模型。