CAREER: Single-Molecule Study of Nucleic Acid Conformational Dynamics in Telomere

职业：端粒核酸构象动力学的单分子研究

• 批准号: 2338902
• 负责人: Hui-Ting Lee
• 金额: $ 97.2万
• 依托单位: University of Alabama at Birmingham
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-02-01 至 2029-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2338902&HistoricalAwards=false
• 关键词: CAREER; Single; Molecule; Study; Nucleic

Telomeres protect the ends of chromosomes. They are composed of special repetitive DNA, proteins, and the RNA version of the DNA. Telomeric DNA can fold into structures called G-quadruplexes, form lariat structures called telomere loops, and form R-loops when it is transcribed into RNA. Solving the unknown structural dynamics during telomere looping and transcription will provide new insights to how telomeres carry out their function of protecting chromosomes, and how aging happens at the cellular level. This project will use a recently developed model telomere DNA system to study DNA structure in real-time, and how DNA structures control telomere looping and transcription. This project will also develop a continuous program that allows high school teachers and students to work in research and teaching laboratories. The inclusion of high school teachers and students is expected to improve the science education on the fundamentals about DNA and RNA. Overall, this project will advance the knowledge of how DNA and RNA works in telomere and propagate basic knowledge of DNA and RNA functions to high school teachers and students. The research goal of this project is to understand how the slow folding process of non-canonical secondary structures affects telomere maintenance. This project seeks to characterize the conformational dynamics of telomeric DNA, RNA, and DNA-RNA hybrid complexes by combining single-molecular Förster resonance energy transfer microscopy (smFRET), calorimetry, and CD spectroscopy. This research will advance mechanism-based understanding of (a) how conformational dynamics in the telomeric DNA and RNA affect the formation of telomere loop and end protection, and (b) how telomeric and non-telomeric sequences self-regulate their transcription. The research goals of this project will be addressed by the following specific objectives: (1) Quantify the sequence dependence of G-quadruplex (G4) refolding kinetics; (2) Characterize the structural and conformational dynamics induced by telomeric repeat-binding factor 2 (TRF2); and (3) Measure the correlation between telomere transcription and TRF2-induced structural changes. The preliminary data from the PI’s laboratory leads to a hypothesis that the slow process of forming a stable G4 in telomeric G-rich ssDNA serves as a biological timer. The outcome of this work will advance our knowledge about how telomeres respond to regulatory factors and the environment, help to reveal the molecular mechanisms of telomere maintenance, and lead to better understanding of cell cycle regulation. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Established Program to Stimulate Competitive Research (EPSCoR).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

端粒保护染色体的末端。它们由特殊的重复DNA、蛋白质和DNA的RNA版本组成。端粒DNA可以折叠成称为g -四联体的结构，形成称为端粒环的分支结构，并在转录成RNA时形成r环。解决端粒环化和转录过程中未知的结构动力学将为端粒如何执行其保护染色体的功能以及衰老如何在细胞水平上发生提供新的见解。本项目将使用最近开发的模型端粒DNA系统实时研究DNA结构，以及DNA结构如何控制端粒环和转录。该项目还将开发一个连续的项目，允许高中教师和学生在研究和教学实验室工作。高中教师和学生的参与有望改善有关DNA和RNA基础知识的科学教育。总体而言，该项目将推进DNA和RNA在端粒中的工作原理，并向高中教师和学生传播DNA和RNA功能的基础知识。本项目的研究目的是了解非规范二级结构的缓慢折叠过程如何影响端粒的维持。本项目旨在通过结合单分子Förster共振能量转移显微镜（smFRET）、量热法和CD光谱学来表征端粒DNA、RNA和DNA-RNA杂交复合物的构象动力学。本研究将促进基于机制的理解(a)端粒DNA和RNA的构象动力学如何影响端粒环和末端保护的形成，以及(b)端粒和非端粒序列如何自我调节其转录。本项目的研究目标将通过以下具体目标来实现：(1)量化g -四重体（G4）重折叠动力学的序列依赖性；(2)表征端粒重复结合因子2 （TRF2）诱导的结构和构象动力学；(3)测量端粒转录与trf2诱导的结构变化之间的相关性。来自PI实验室的初步数据导致了一个假设，即在富含g的端粒ssDNA中形成稳定G4的缓慢过程起到了生物计时器的作用。这项工作的结果将促进我们对端粒如何响应调节因子和环境的认识，有助于揭示端粒维持的分子机制，并有助于更好地理解细胞周期调节。该项目由分子和细胞生物科学部的分子生物物理集群和促进竞争研究的既定计划（EPSCoR）共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。