单分子水平研究G-四链体RNA的结构动力学及其与eIF4A的相互作用

Guanine (G)-rich sequences in DNA/RNA can fold into non-canonical G-quadruplex (G4) structures by Hoogsteen hydrogen bonding. Although G4 RNA structural dynamics and its interaction with G4 RNA helicases are well known to play essential regulatory roles in various life processes. Unfortunately, the microscopic molecular mechanisms of the dynamic folding and unfolding of G4 RNA structure as well as its interaction mechanism with G4 RNA helicases unexplored yet. Again, it warrants to mentioning that the advancements in single-molecule imaging techniques provide efficient methods for exploring structural dynamics and macromolecular interactions in real time. Therefore, the present proposal was designed with aims to study the folding and unfolding mechanism of G4 RNA, effect of internal as well as external stresses on structural dynamics of G4 RNA, the process of eIF4A binding and unwinding of G4 RNA, and the impact of G4 RNA on eIF4A structure and behaviors by selecting G4 RNA and eIF4A helicase, which are known to related with cancer in animal and plant response to stress, by using single-molecule fluorescence resonance energy transfer (smFRET) method. The expected outcomes of this project will not only provide a basis for understanding the regulatory mechanism of G4 RNA during life processes but also will provide a theoretical foundation for the treatment of cancer as well as in the development of plant varieties resistant to abiotic stresses.

富含鸟嘌呤的DNA/RNA序列可以通过Hoogsteen氢键折叠成非传统的G-四链体（简称G4）结构。虽然G4 RNA的动态结构及其与G4 RNA解旋酶的相互作用在生命过程中发挥着重要的调节作用，但是G4 RNA的动态折叠和打开过程以及G4 RNA与解旋酶相互作用的微观分子机理仍未得到阐明。单分子技术的发展为实时研究生物大分子结构变化和相互作用提供了有效方法。本项目拟选取与动物癌症发生和植物抗逆过程相关的G4 RNA和eIF4A解旋酶为研究对象，利用单分子荧光共振能量转移（smFRET）技术，深入研究G4 RNA折叠和去折叠的路径，内外条件对G4 RNA结构动力学的影响，eIF4A结合并解旋G4 RNA的分子过程，以及G4 RNA对eIF4A结构和行为的影响。本项目的实施和完成将为理解G4 RNA对生命过程的调节机制提供科学依据，也为癌症治疗和抵抗非生物逆境胁迫植物品种的设计提供理论参考。

细胞内，富含鸟嘌呤(Guanine, G)的DNA/RNA序列可以折叠形成G-四链体结构(G-quadruplex, G4)。该结构在许多重要生物过程中发挥着重要的调节功能，与人类健康和植物生长发育密切相关。目前，绝大多数关于G4的研究都集中在DNA G4 (dG4)上。虽然人们已经证明了部分RNA G4 (rG4)的功能，但是rG4的折叠和打开动态过程及折叠路径尚不清楚。解旋酶在rG4结构调控中发挥着关键作用，但解旋酶打开rG4的微观分子机理仍未得到阐明。本项目选取与动物癌症发生和植物抗逆过程相关的rG4s和eIF4A解旋酶为研究对象，尝试回答以上问题。本研究主要利用本实验室建立的单分子荧光共振能量转移(smFRET)技术，在单分子水平深入研究rG4折叠和去折叠的路径，以及eIF4A与rG4的相互作用。首先，我们选择四种典型的rG4结构(两层和三层各两个)，利用圆二色谱法确定了G4结构的存在。然后，通过改变溶液中K+终浓度研究K+诱导rG4折叠的过程，利用多峰高斯拟合和隐马尔可夫模型得到了四个折叠状态。接着，利用rG4稳定剂和底物突变，以及dG4对照的方法，确定了G-发卡(G-hairpin)和G-三链体(G-triplex)是rG4动态折叠的中间状态，揭示了rG4的折叠路径。通过eIF4A与rG4底物亲和力测定证明rG4是eIF4A的天然底物，并通过smFRET证明eIF4A确实能够解旋rG4结构。本项目的研究结果为理解rG4结构调节生命过程机制提供科学依据，也为癌症治疗和抵抗非生物逆境胁迫植物品种的设计提供理论参考。