CAREER: High-throughput multi-axial tension-inducing DNA origami device for investigating mechanosensitive signaling pathways and protein structures under defined tension

职业：高通量多轴张力诱导 DNA 折纸装置，用于研究限定张力下的机械敏感信号通路和蛋白质结构

• 批准号: 2341002
• 负责人: Rizal Hariadi
• 金额: $ 110万
• 依托单位: Arizona State University
• 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=2341002&HistoricalAwards=false
• 关键词: CAREER; throughput; multi; axial; tension

Minuscule mechanical forces within us regulate the functions of mechanosensitive proteins, which subsequently control how cells act and react. By studying integrins, the tiny proteins that serve as cellular anchors and sensors for the mechanical cues from their environments, new rules governing how cells translate mechanical forces they feel into decisions regarding their behavior can be uncovered. This insight will advance the understanding of living systems and has enormous potential in understanding diseases like cancer, where the stickiness and mechanical properties of cells play key roles. Beyond its scientific merit, the research's impact will stretch beyond the laboratory. An innovative, hands-on course that immerses undergraduate students in Biological Physics research, fostering the next wave of scientists, will be designed. Additionally, artists will help create science-inspired cartoons, making complex scientific concepts accessible and thrilling for children of all ages.This project will develop a novel approach to understanding the mechanotransduction pathways and structural biology of integrin proteins, a critical factor for cell adhesion and signaling. At the heart of this project is the Multi-Axial Entropic Spring Tweezers along Ring-shaped Origami (MAESTRO), a low-cost, high-throughput technology for applying controlled mechanical tensions to individual integrins from multiple directions. MAESTRO leverages the entropic elasticity of single-stranded DNA to generate mechanical tension, the exquisite positional control of DNA origami to control the directions of applied tensions, and device miniaturization to reduce cost and increase throughput. To validate the performance of MAESTRO, the tension-inducing tool will be benchmarked by assessing the kinetics of Holliday junctions under mechanical tension. Measurements will be compared with published data from optical tweezer experiments and theoretical predictions derived from the Boltzmann distribution. Advanced single-molecule methods, including confocal single-molecule Fluorescence Resonance Energy Transfer (smFRET) and total internal reflection microscopy, will be used to dissect the intricate signaling of integrins in response to these multi-directional mechanical stimuli. Finally, cryo-electron microscopy will be used to visualize the tension-induced state changes of integrins, offering structural insights into their signaling roles.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.

美国内部的微小机械力调节机械敏感蛋白的功能，随后控制细胞的作用和反应。通过研究整联蛋白，从其环境中用作机械提示的细胞锚和传感器的微小蛋白质，可以发现细胞如何将其感受到有关其行为决定的机械力转化为对其行为的决定的新规则。这种见解将提高人们对生活系统的理解，并在理解诸如癌症之类的疾病（细胞的粘性和机械特性起着关键作用）中具有巨大的潜力。除了其科学优点外，研究的影响还将超越实验室。将设计一种创新的动手动手课程，将本科生沉浸在生物物理学研究中，促进下一波科学家。此外，艺术家将有助于创建科学启发的动画片，使各个年龄段的儿童都可以访问复杂的科学概念并激动人心。该项目将开发一种新的方法来理解整联蛋白蛋白的机械传导途径和结构生物学，这是细胞粘附和信号传导的关键因素。该项目的核心是沿环形折纸（Maestro）的多轴熵弹簧镊子，这是一种低成本的高通量技术，用于从多个方向将受控的机械张力应用于单个整合素。 Maestro利用单链DNA的熵弹性产生机械张力，DNA折纸的精美位置控制以控制施加的张力的方向以及设备微型化，以降低成本并增加吞吐量。为了验证大师的表现，通过评估机械张力下的霍利迪连接的动力学来基准诱导张力的工具。测量结果将与来自光学镊子实验的已发布数据和从玻尔兹曼分布得出的理论预测进行比较。晚期单分子方法，包括共焦单分子荧光共振能量转移（SMFRET）和总内反射显微镜，用于剖析整合素的复杂信号，以响应这些多向机械刺激。最后，冷冻电子显微镜将用于可视化整联蛋白的张力诱导的状态变化，从而为其信号传导角色提供结构性见解。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识分子优点和更广泛的影响审查标准来评估的值得支持的。