【作者】 张凌；

【导师】 李景虹；

【作者基本信息】 清华大学 ， 化学， 2017， 博士

【摘要】 直接分析单个分子的微区结构,构象变化,动态行为,相互作用的单分子分析技术,能为理解分子的结构与功能机制提供更加丰富的信息。纳米通道作为一种有潜力的第三代基因测序技术,为分子生物学和纳米科学提供了实时、快速的单分子检测平台。本论文以α-溶血素蛋白(α-Hemolysin)的纳米级通道结构为基础,利用其对单分子精细结构高灵敏的分辨能力,提出了高特异性的分子识别策略,构建了新型单分子分析方法和传感器,应用于癌症标志物蛋白的高选择性富集与检测,小分子药物的作用机制与热力学分析,以及纳米簇结构的高灵敏识别。本文的具体研究内容如下:基于“邻近结合”核酸引发的DNA链置换反应,发展了一种高选择性检测癌症标志物蛋白血小板生长因子(PDGF)的新方法。两条具有特异性核酸适配体(Aptamer)和碱基互补序列的DNA链作为探针,同时识别一个目标蛋白引发核酸探针的“邻近结合”和DNA的链置换反应,释放的单链DNA被纳米通道识别并输出信号,实现目标物蛋白的定量分析。该方法选择性好,快速便捷,无需标记、化学修饰和信号放大,可运用于血清样品检测。在单分子尺度下对人端粒DNA序列与药物pyridostatin的相互作用进行了探究,以α-溶血素前厅空腔捕获并区分G-四联体结构。通过分析单分子信号阻断时间的不同,证明了药物对G-四联体结构机械稳定性的增强效应。发现了“两段式”单分子信号,通过自由能的分析,证明了K~+和药物对G-四联体稳定的协同作用。同时,发展了快速、非标记热力学计算方法,无需改变受体和配体分子浓度,具有很好的重复性和准确性,有希望应用于小分子抗癌药物的筛选。在单分子层次上分析了包括杂多酸盐和金纳米簇在内的五种不同尺寸结构的纳米簇,通过结构相关的二维阻断时间-阻断电流谱图和归一化捕获速率,可实现多种不同纳米簇结构的实时识别和区分。相比传统分析方法,纳米通道对样品浓度检测的灵敏度高出3个数量级。通过分子动力学模拟,分析了纳米簇穿过纳米通道的动态过程和引起的离子电流阻断,理论模拟结果进一步证明了实验结果与纳米簇结构的关联。纳米通道分析平台可为纳米簇结构的分析表征提供新的工具。更多还原

【Abstract】 Single-molecule analytical techniques that analyze the structure,confomatinal change,dynamic and interaction of the analytes at single-molecule level can provide more information of molecular structure and their function or mechanism.As a potential technique for the third generation gene sequencing,nanopore has provided a rapid and real-time single-molecular analytical platform for molecular biology and nanoscience.Based on the nanostructure of a biological nanoporeα-Hemolysin(α-HL)and its great ability to sense and distinguish single-molecular structures,this thesis has proposed strategies for highly specific molecule recognition and developed single-molecular analytical methods and sensors for the cancer-related biomarker detection,small molecule drug mechanism and thermodynamic analysis,and nanocluster structure identification.The details are as follows:A highly selective nanopore proximity bioassay for protein biomarker platelet-derived growth factor B-chain(PDGF-BB)detection was presented based on a proximity binding-induced DNA strand displacement strategy.An individual target protein was recognized by two specific oligonucleotide aptamers which were linked with complementary sequences as probes.The protein recognition induced the proximity of two probes and an output DNA was released and translocated throughα-HL nanopore with an output signal,allowing the quantification of the target protein.This rapid and label-free bioassay had great selectivity without the need of chemical modification or signal amplification,and could be applied in serum samples.Human telomere sequence interactions with a small molecule drug pyridostatin(PDS)were analyzed in single-molecule level.Different telomeric DNA G-quadruplex structures were captured and discriminated by the vestibule ofα-HL nanopore.The potent stabilization effect of drug on G-quadruplex structure was demonstrated by analysis of the unraveling time of G-quadruplexes.Signature two-level electronic blocks were discovered.The translocation studies and the free-energy analysis demonstrated a coordinated effect of K~+and drug on G-quadruplex stabilization.Additionally,the nanopore platform permits the efficient and accurate determination of drug affinity constants without the requirement for labeling,amplification,or ligand/receptor titration,possessing great potential for the design and screen of anticancer drugs.Multiple nanoclusters including kinds of polyoxometalates and Au nanocluster were analysed in single-molecule level viaα-HL nanopore,by providing structure-dependent 2D dwell time-current blockage spectrums and normalized capture rates for the cluster translocation events.Theα-hemolysin ion channel permits the discrimination of nanocluster structures with atomic precision,and shows 1000-fold higher sensitivity in analyte concentration than other classical methods.Molecular dynamics(MD)simulations furtherly revealed the nanocluster translocation dynamics and related the experimental result with the nanocluster structure.The nanopore platform provides a novel powerful tool for nanocluster characterization.更多还原