基于邻近标记技术的蛋白标记方法用于活细胞内G4相关蛋白的钓取及其功能研究

G4相关蛋白与G4核酸之间的动态相互作用是揭示G4核酸生命调控功能的重要切入点，但是这种动态相互作用常常难以在实验条件下被捕捉，因此这也是G4功能研究的主要难点。目前的G4相关蛋白鉴定方法还具有很大的完善空间。为了深入研究G4与其相关蛋白的相互作用，本项目拟开发两种基于邻近标记技术的G4相关蛋白标记方法，用于活细胞内G4相关蛋白的钓取及鉴定。第一种，利用G4靶向的短肽RHAU23与生物素连接酶的融合蛋白对细胞中的G4进行识别并对邻近的蛋白生物素化标记；第二种，利用G4与血红素复合物的拟过氧化物酶活性对活细胞中G4附近的蛋白进行催化标记。本项目还将从所鉴定的G4相关蛋白中选取对细胞正常生理调控及癌症发生发展相关的G4解旋蛋白，对其细胞内的作用靶点及其对靶基因表达的调控功能进行研究。本项目的开展有望发现更多新的G4相关蛋白，有助于进一步揭示G4在正常生理活动及疾病发生和治疗过程中的重要功能。

The interactions between G-quadruplex(G4) and G4 related proteins are dynamically changed, which declares its significant aspect to reveal the biological function of G4. But these dynamic interactions are often difficult to be captured under experimental conditions, that makes it the main difficulty in the study of G4’s biological function. There is still much we can do to develop the identified method of G4 related proteins. For the purpose to study the interactions between G4 and its related proteins deeply, two new strategies were established to pull-down intracellular G4 related proteins in this project, which mainly based on enzyme-catalyzed proximity labeling. The first one is based on the fusion protein of a G4 targeting short peptide RHAU23 and a biotin ligase, which can specifically recognize G4 in vivo and labels the adjacent proteins with biotin. The second is based on G4-hemin peroxidase-mimicking activity, which can catalyze the covalent labeling of proximal proteins. We also conduct an in-depth study on G4 helicase which is related to normal cell regulation and cancer development, to reveal its target sites and regulatory functions in vivo. The establishment of these new methods are expected to find more G4 related proteins, which will help to further reveal the G4’s important roles in normal physiological activities and in the development and therapy of diseases.