用单分子方法研究G-四链体DNA结构稳定性和解旋机制

染色体中存在一些富含鸟嘌呤的区域，鸟嘌呤在hoogsteen氢键和一价阳离子作用下堆积成稳定的G－四链体（G4）。人类染色体端粒带有～200nt的GGGTTAGGGTTAGGGTTAGGG重复序列，通过折叠成稳定的G4结构抑制端粒酶工作，来调控端粒延伸。 G4序列也存在于基因启动子区域，通过折叠／去折叠的转换对基因转录表达进行调控。G4结构由于对端粒和基因表达的调控作用，已经成为十分重要的抗癌药物设计靶点。遗憾的是，人们对G4折叠路径，结构热涨落等物理性质还缺乏深入了解，对G4结构解旋机理的研究更是刚刚起步。我们将借助先进的单分子生物物理手段（包括磁镊和单分子荧光共振能量转移）来研究：(1) G4 DNA稳定性的物理学基础，包括折叠路径、高级结构形成机制、序列对结构的影响等；(2) G4 解旋微观机制，包括解旋酶聚集状态、解旋方向、是否存在中间过程、解旋速率、解旋效率、步长、持续解旋距离等

In the chromosome, there are some guanine rich regions. These guanines form G-tetrad by the hoogsteen hydrogen bonds, and then fold into G-quadruplex stabilized by monovalent ion such as K+, Na+. At the end of human chromosome telomere, there is a single strand repeated sequence GGGTTAGGGTTAGGGTTAGGG of ~200nt, which is able to fold into G-quadruplex and block the telomerase elongation of telomere DNA. Besides, G4 sequences were also identified to exist inside genome, especially in promoter region. By switching between folded and unfolded states, those sequences are able to regulate gene transcription and expression. However, until now, there is a lack of deep understanding of G-quadruplex physical properties such as folding pathway and structural fluctuations. The investigation of G-quadruplex unwinding mechanism by helicase has just started recently. We are going to use single molecule methods including magnetic tweezers and single molecule FRET to study: (1) the physical basis for G-quadruplex stability: folding pathway, higher structure formation mechanism and the effects of sequence on the structure； (2) G-quadruplex unwinding mechanism, including helicase assembly state, G-quadruplex intermediate state, unwinding velocity, efficiency, step size, processivity. The answers to those questions will provide deep understanding of the biological functions of G-quadruplex, and benefit the G-quadruplex targeted anti-cancer drug design.