用单分子方法研究G-四链体DNA/RNA解旋酶的作用机理

G-quadruplexes (G4) are highly compact nucleic acids structures formed by Guanine-rich DNA or RNA that fold into a four-stranded conformation. G4 DNA is distributed in gene promoters and chromosome telomere regions while G4 RNA is in messenger RNA and telomere RNA produced by gene and telomere transcription. G4 structures play important roles in the maintenance of telomere stability and regulation of gene expression. Examining G4 structures and their regulation helps our further understanding of cancer and aging. In cells, G4 structures are controlled timely and spatially by a series of highly specific helicases. It is challenging to study G4 unfolding mechanisms by these helicases and the relevant studies are at the very early stage. In this project, we plan to use an advanced single molecule approach, especially the conjugation of magnetic tweezers and single molecule FRET, to study the detailed unfolding mechanisms of several important G4 DNA, RNA helicases. Questions will be examined include how those helicases recognize G4 structures specifically, how helicases translocate on G4, what their oligomeric states and the intermediates of G4 unfolding are, and how ATP functions in regulation of molecular motors et al. We will also compare the differences between G4 DNA and RNA unfolding. Our study will help further understanding of the physical properties and biological functions of G4, and benefit the G4 targeted anti-cancer drug design.

G-四链体是由富含鸟嘌呤的DNA、RNA序列折叠成的具有四条链的高级致密核酸结构。G4 DNA广泛存在于基因启动子和染色体端粒，G4 RNA存在于转录产生的信使RNA和端粒RNA，对端粒的稳定和基因表达起重要调控作用。对G4调控机理的认识将有助于对癌症和衰老现象的理解。在细胞内，高度稳定的G4结构是如何由特异的解旋酶在特定时空状态下来调控的，是一个极具挑战性的研究。G4解旋酶调控的分子机理方面的研究才刚刚起步。在本项目中，我们将借助先进的单分子生物物理手段，特别结合我们发展的单分子磁镊和单分子荧光耦合技术，研究几类重要G4 DNA、RNA解旋酶的微观工作机制，包括酶如何识别G4，酶的移动方向、多聚状态、G4解旋中间过程、ATP驱动的分子马达调控等，并对比G4 DNA和RNA在解旋机理上的差异。本项目的实施，对理解G4结构的物理学基础和生物学功能，推动以G4为靶点的抗癌药物设计具有重要意义。

G-四链体是由富含鸟嘌呤的DNA、RNA序列折叠成的具有四条链的高级致密核酸结构。G4 DNA广泛存在于基因启动子和染色体端粒，G4 RNA存在于转录产生的信使RNA和端粒RNA，对端粒的稳定和基因表达起重要调控作用。对G4调控机理的认识将有助于对癌症和衰老现象的理解。在细胞内，高度稳定的G4结构是如何由特异的解旋酶在特定时空状态下来调控的，是一个极具挑战性的研究。G4解旋酶调控的分子机理方面的研究才刚刚起步。.在本项目中，我们借助先进的单分子生物物理手段，特别结合我们发展的单分子磁镊和单分子荧光耦合技术，用单分子方法研究G4 DNA结构的折叠和去折叠动力学，证实了G4 DNA 折叠中间态G-triplex和G-hairpin的存在，最终提出人类端粒DNA结构的多路径折叠机制；发现同一个ScPif1蛋白在解旋G4后坐在ss/dsDNA岔口处，在G4反复形成的过程中，重复性的解旋G4结构。同时发现，Pif1先后两步打开G4结构，实验证明在此过程中存在稳定的中间态-G3结构。这种重复解旋活性有利于Pif1打开后续形成的G4结构，修复停滞的复制叉；系统分析了Pif1解旋酶解旋G4作用特点；进一步从原子尺度上阐释了Pif1家族解旋酶的解旋模式，并深入发掘出了经典物种酿酒酵母ScPif1中参与解旋调控的全新结构域；提出了RHAU解旋G4结构的机制为“主动-被动”解旋机制，即RHAU结合G4后可以使G4结构变得不稳定，之后通过消耗ATP的主动解旋过程使得G4结构彻底打开。.本项目的实施，对理解G4结构的物理学基础和生物学功能，推动以G4为靶点的抗癌药物设计具有重要意义。