Mechanical forces in nanoscale biology: From hemostasis to single-molecule centrifugation

纳米生物学中的机械力：从止血到单分子离心

• 批准号: 10631055
• 负责人: Wesley Philip Wong
• 金额: $ 48.68万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10631055
• 关键词: Acceleration; Address; Adhesions; Adhesives; Area; Behavior; Biochemical; Biological Assay; Biology; Blood Coagulation Disorders; Cells; Centrifugation; Communicable Diseases; Complex; DNA; Democracy; Development; Devices; Disease; Genomics; Growth; Hair Cells; Hearing; Hemostatic function; Immune response; Immunology; Leukocytes; Life; Link; Malignant Neoplasms; Measurement; Measures; Mechanics; Methods; Microscope; Molecular; Movement; Organism; Pathway interactions; Play; Process; Property; Protein Conformation; Proteins; Regulation; Research Personnel; Robotics; Role; Shapes; Spectrum Analysis; Structure; Technology; Tissues; centrifuge force microscope; deafness; driving force; high throughput screening; insight; instrument; instrumentation; interest; mechanical force; mechanotransduction; nanoscale; nanoswitch; response; screening; single molecule; technology development; tool; von Willebrand Factor

Abstract Mechanical forces play key roles throughout biology, from governing the adhesion of leukocytes in the immune response, to determining cell fate and directing tissue formation. This field of mechanobiology is providing vital insights into conditions such as bleeding disorders, cancer, and infectious diseases, where it is becoming clear that conventional biochemical and genomic characterizations are not sufficient to understand the rich behavior of living systems or how they fail. Rather, we must uncover how force changes the structure and function of molecules, triggering mechanotransduction pathways to modify cell responses. Technological developments that enable precise manipulation of single molecules and cells have been a driving force in the development of the field, but growth has been impeded by both limited access to such technologies and by constraints in their capabilities, which has restricted the types of scientific questions that can be addressed. To overcome these challenges, we will develop approaches in mechanobiology that will (i) open up new areas of study through the introduction of new capabilities, and (ii) democratize single-molecule and nanoscale methods so that all biomedical researchers can make discoveries using these powerful tools. We will continue to develop instruments such as the Centrifuge Force Microscope, a miniature microscope that fits into a benchtop centrifuge to enable even non-specialists to perform high-throughput single-molecule force measurements, and nanoscale devices such as programmable DNA nanoswitches. We will develop DNA nanoswitch calipers, a tool capable of measuring distances on single-molecules with angstrom-level precision to enable single-molecule protein identification and shape determination. We will also develop Functional Interaction-based Nanoswitch Discovery (FIND), a high-throughput screening assay based not on traditional robotics, but on molecular devices that bring together molecular components to analyze and screen for interactions of interest. FIND will enable screening of complex modes of action to find compounds that activate a specific downstream pathway or allosterically stabilize a particular protein conformation. We will apply our nanoscale approaches to answer key open questions in mechanobiology. For example, to uncover the mechanical regulation of hemostasis we will use single-molecule methods to study the force- regulated enzymatic cleavage of von Willebrand factor, and the flow-induced elongation and activation of its adhesive function. We will also investigate the molecular basis of hearing and deafness by using single- molecule force spectroscopy to probe the properties of the hair cell tip link, and combine this approach with single-channel conductance measurements to simultaneously measure the force required to open mechanotransduction channels and the molecular movements that underlie channel gating. Overall, these efforts should firmly establish force as a key parameter for understanding the basic processes of life, and provide a new handle for both understanding—and treating—disease.

摘要 机械力在整个生物学中起着关键作用，从控制免疫系统中白细胞的粘附， 反应，决定细胞命运和指导组织形成。机械生物学的这一领域提供了 对出血性疾病、癌症和传染病等疾病的深入了解， 传统的生物化学和基因组特征不足以理解丰富的行为， 生命系统或它们是如何失败的。相反，我们必须揭示力是如何改变结构和功能的。 分子，触发机械转导途径以改变细胞反应。技术发展 能够精确操纵单个分子和细胞的技术已经成为开发 但由于获得这些技术的机会有限， 这限制了可以解决的科学问题的类型。 为了克服这些挑战，我们将开发机械生物学的方法，这些方法将（i）开辟新的领域 通过引入新的能力，研究的，（ii）民主化的单分子和纳米 所有生物医学研究人员都可以使用这些强大的工具进行发现。我们将继续 开发仪器，如微力显微镜，微型显微镜，适合在一个 台式离心机，使即使是非专业人员也能进行高通量单分子力 测量和纳米级器件，如可编程DNA纳米开关。我们将开发DNA 纳米开关卡尺，能够以埃级精度测量单分子距离的工具 以实现单分子蛋白质鉴定和形状确定。我们还将开发功能 基于相互作用的纳米开关发现（FIND），一种不基于传统纳米开关的高通量筛选试验。 机器人技术，而是在分子设备上，将分子成分聚集在一起进行分析和筛选， 兴趣的互动。FIND将能够筛选复杂的作用模式，以找到激活 特定的下游途径或变构稳定特定的蛋白质构象。 我们将应用我们的纳米方法来回答机械生物学中的关键开放问题。例如以 揭示止血的机械调节，我们将使用单分子方法来研究力- 调节血管性血友病因子的酶促裂解，以及其流动诱导的延伸和激活， 粘合功能我们还将研究听力和耳聋的分子基础，通过使用单- 分子力光谱探测毛细胞尖端连接的特性，并将这种方法与联合收割机结合起来， 单通道电导测量，同时测量打开所需的力 机械转导通道和作为通道门控基础的分子运动。总的来说，这些 努力将力牢固地确立为理解生命基本过程的关键参数， 为理解和治疗疾病提供了新的方法。

项目成果

Force Spectroscopy and Beyond: Innovations and Opportunities.

• DOI: 10.1016/j.bpj.2018.10.021
• 发表时间: 2018-12-18
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Nathwani B;Shih WM;Wong WP
• 通讯作者: Wong WP

Single-molecule mechanical fingerprinting with DNA nanoswitch calipers.

• DOI: 10.1038/s41565-021-00979-0
• 发表时间: 2021-12
• 期刊: Nature nanotechnology
• 影响因子: 38.3
• 作者: Shrestha P;Yang D;Tomov TE;MacDonald JI;Ward A;Bergal HT;Krieg E;Cabi S;Luo Y;Nathwani B;Johnson-Buck A;Shih WM;Wong WP
• 通讯作者: Wong WP

Mapping Single-Molecule Protein Complexes in 3D with DNA Nanoswitch Calipers.

• DOI: 10.1021/jacs.3c10262
• 发表时间: 2023-12-27
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Shrestha, Prakash;Yang, Darren;Ward, Andrew;Shih, William M.;Wong, Wesley P.
• 通讯作者: Wong, Wesley P.