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.

我们体内微小的机械力调节机械敏感蛋白质的功能，这些蛋白质随后控制细胞的行为和反应。通过研究整合素，可以发现细胞如何将感受到的机械力转化为有关其行为的决定的新规则。整合素是一种微小的蛋白质，充当细胞锚和来自环境的机械信号的传感器。这种洞察力将促进对生命系统的理解，并在理解癌症等疾病方面具有巨大的潜力，在这些疾病中，细胞的粘性和机械性能起着关键作用。除了它的科学价值，这项研究的影响将延伸到实验室之外。将设计一门创新的动手课程，让本科生沉浸在生物物理研究中，培养下一波科学家。此外，艺术家们将帮助创作以科学为灵感的卡通片，使复杂的科学概念变得容易理解，并为所有年龄段的儿童带来刺激。这个项目将开发一种新的方法来理解整合素蛋白质的机械转导途径和结构生物学，整合素蛋白质是细胞黏附和信号传递的关键因素。该项目的核心是沿环形折纸(MAESTRO)的多轴熵弹簧钳(MAESTRO)，这是一种低成本、高通量的技术，可以从多个方向对单个整合素施加受控的机械张力。Maestro利用单链DNA的熵弹性来产生机械张力，利用DNA折纸的精密位置控制来控制施加张力的方向，并利用设备微型化来降低成本和增加产量。为了验证Maestro的性能，将通过评估Holliday结在机械拉伸下的动力学来对张力诱导工具进行基准测试。测量结果将与已发表的光镊子实验数据和由玻尔兹曼分布得出的理论预测进行比较。先进的单分子方法，包括共聚焦单分子荧光共振能量转移(SmFRET)和全内反射显微镜，将被用来剖析整合素响应这些多方向机械刺激的复杂信号。最后，冷冻电子显微镜将被用来可视化张力诱导的整合素的状态变化，提供对其信号角色的结构洞察。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。