针对蛋白质-核酸相互作用研究的单分子荧光偏振分析方法学与技术

Protein-DNA binding analysis is an important mean for the understanding of biological processes and functions and the disease development. All cells from three organism kingdoms utilize the interactions of multiple proteins with the same DNA fragment to finely regulate gene expression and silence. Therefore, it is extremely important to develop analytical technologies to study the interactions of multiple proteins with DNA. However, there is a technology gap. We propose a project to develop a novel single-molecule fluorescence polarization analysis and to study the suggested multiple protein-DNA interactions. The proposed technology is very different from the known single-molecule fluorescence resonance energy transfer analysis, and it does not require the distance changes involved in protein-DNA interactions, and may indicate the stoichiometry of multiple-protein binding to same DNA molecules by fluorescence polarization signaling. We will study the simultaneous binding of RecA protein and Single Strand Binding (SSB) protein to single stranded DNA to show the unique capacity of single-molecule fluorescence polarization characterizing the protein-DNA interactions. Furthermore, we will clarify the mechanisms on the cooperative assembly and disassembly of RecA on ssDNA, and the role of ATP regulation played in the RecA-ssDNA interaction. We will also identify the critical intermediates of RecA-mediated strand exchange, which is core reaction in recombination repair maintaining genome stability. The study will further our understanding of biologically important recombination repair and the cancer incidence. Moreover, we will extend our technology to study the role of nonspecific interaction played in the specific recognition of protein against target DNA sites, and to characterize the conformational change involved in high-affinity and highly selective protein-aptamer binding.

蛋白质与核酸的相互作用分析是理解生命过程和疾病发生的重要手段之一。各种生物细胞正是通过多个蛋白质结合于同一个DNA分子这一方式精细地调控基因表达与沉默。因此，研究多个蛋白质分子同时与一个DNA片段的结合极其重要。我们提出发展新颖的单分子荧光偏振分析技术和方法。与已有的单分子荧光共振能量转移分析不同的是，这种单分子分析毋需依赖相互作用过程中发生距离变化，可表征多种蛋白质与DNA的结合计量学。我们将通过研究RecA蛋白、SSB蛋白与ssDNA的同时相互作用，展示单分子荧光偏振分析的独特性能。在方法发展基础上，揭示RecA在ssDNA上的协同组装及去协同作用机理，鉴定介导链交换反应的关键RecA-ssDNA组装体，有助于人们对重组修复与癌症发生发展的认识。我们将进一步利用单分子荧光偏振分析，探讨蛋白质-核酸相互作用中的非特异性作用和特异性识别机制，以及核酸适配体与靶蛋白作用的动态构象变化。

依托本项目，我们建立了新颖的单分子荧光偏振分析技术与方法，并将其成功应用于多种蛋白质与（脱氧）核酸以及其他功能核酸分子（适配体、DNAzyme等）与靶标分子的相互作用研究。我们利用发展的新方法揭示了大肠杆菌同源重组酶RecA在单链DNA（ssDNA）上协同组装的机理，并提出了ATP水解紧密调控RecA组装形成低密度不饱和RecA核蛋白丝促进DNA链交换及同源重组修复的模型。我们还利用这种单分子技术考察了人源hAGO2蛋白与DNA及MicroRNA（miRNA）的相互作用，揭示了一种新的hAGO2蛋白依赖的miRNA在细胞核内正向调控基因表达的分子机制。基于G碱基与荧光染料作用增强荧光偏振响应的原理，我们发展了新型的荧光偏振传感器可以用于快速高灵敏地检测Pb2+、Ag+、半胱氨酸、谷胱甘肽。此外，我们还发展了适配体的多构象分析技术，能够用于表征适配体与靶标分子结合前后的构象变化，从而揭示其相互作用的结构基础，并在此基础上提出了一种基于多重相互作用增强适配体亲和力的方法。在本项目的资助下，已发表sci论文49篇，其中影响影子大于5的21篇，包括12篇Analytical Chemistry，2篇Trends in Analytical Chemistry，2篇Biosensors Bioelectronics；申请专利12项，已授权2项；培养博士研究生6名。