An Activity-Based Biomolecule Labeling Platform for the Imaging of Cells and Tissues Under Oxidative Stress

基于活性的生物分子标记平台，用于氧化应激下细胞和组织的成像

• 批准号: 10283664
• 负责人: Marco Messina
• 金额: $ 9.15万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10283664
• 关键词: Alzheimer' s Disease; Biological; Biological Models; Cardiovascular Diseases; Cell Communication; Cell Death; Cell Line; Cell physiology; Cells; Chemicals; Coculture Techniques; Collaborations; Complex; Coupled; Data; Detection; Diffuse; Diffusion; Disease; Ethers; Exposure to; Family; Fluorescence Microscopy; Fluorescent Probes; Foundations; Goals; Homeostasis; Hydrogen Peroxide; Hypochlorous Acid; Image; In Vitro; Label; Malignant Neoplasms; Maps; Measurement; Mediating; Metabolic; Microglia; Modeling; Molecular Probes; Monitor; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Noise; Organism; Oxidation-Reduction; Oxidative Stress; Pathology; Phase; Phenols; Play; Polymer Chemistry; Polymers; Production; Property; Proteins; Reaction; Reactive Oxygen Species; Research; Role; Sampling; Signal Transduction; Signaling Molecule; Source; Stress; Structure; Superoxides; Surface; System; Testing; Tissues; Training; Visualization; base; biological systems; cellular imaging; cellular targeting; design; experience; fluorescence imaging; fluorophore; imaging platform; oxidative damage; polymerization; programs; quinone methide; response; small molecule; tool

Project Summary Reactive oxygen species (ROS) are a family of small-molecules in living systems that serve vital roles in both signaling and stress. Hydrogen peroxide (H2O2), superoxide (O2•-), and hypochlorous acid (HOCl), among others, are all examples of ROS that have been traditionally viewed as sources of oxidative stress and damage. Aberrant ROS production contributes to a multitude of pathologies such as neurodegeneration, cancer, and cardiovascular disorders. However, ROS are also critical for maintaining metabolic homeostasis through activation of multiple classes of proteins. This signal-stress dichotomy, coupled with the small and transient nature of ROS, presents a challenge when attempting to decode the complex landscape of cellular redox homeostasis. Fluorescent probes are frequently employed to visualize ROS in living systems through fluorescence microscopy, however these probes are prone to diffusion after ROS detection. This leads to inaccurate determination of ROS localization and poor signal-to-noise responses. As such, there is a need to create probes amenable to the permanent recording of ROS via fluorescence imaging. We hypothesize that activity-based cell-trappable fluorescent probes can be used as a platform to gain further understanding of ROS-mediated inter- and intra- cellular signaling. We propose three specific aims to test this hypothesis. First, we will synthesize fluorophores caged with activity-based triggers and proximal fluoromethyl groups to serve as latent equivalents of quinone methide upon ROS sensing. ROS responsive uncaging will allow for the fluorescent labeling of adjacent biomolecules. Second, we will apply our probes across multiple model live cell lines to monitor ROS fluxes. We will also map cell-to-cell communication mediated by ROS using microglia-neuron co-culture as a biological model. This system will allow us to probe transcellular redox signaling as microglia can be selectively activated in the presence of neurons thereby dispatching H2O2 to nearby neurons. The third aim involves developing a fluorescent polymer amplification strategy to increase signal-to-noise responses of tandem activity-based sensing/labeling probes and will primarily be carried out in the R00 phase. Small- molecule polymer initiators will be caged in a similar manner to the previously described fluorescent probes. After ROS sensing and biomolecule labeling, polymerization will be performed to generate fluorescent polymers from biomolecule surfaces thus enabling signal amplification and visualization. This strategy will be carried over into live cell lines described above. This research fits into the applicant’s goal of establishing a program which uses polymer chemistry to probe fundamental questions in biological systems.

项目概要 活性氧 (ROS) 是生命系统中的一类小分子，为生命活动提供服务 过氧化氢 (H2O2)、超氧化物 (O2•-) 和次氯酸。 酸 (HOCl) 等都是 ROS 的例子，传统上被视为 异常的 ROS 产生会导致多种氧化应激和损伤。 然而，ROS 可能会导致神经退行性疾病、癌症和心血管疾病等疾病。 通过激活多类物质来维持代谢稳态也至关重要 这种信号-应激二分法，加上 ROS 的小且短暂的性质， 在尝试解码细胞氧化还原的复杂景观时提出了挑战 荧光探针经常用于观察生命系统中的 ROS。 通过荧光显微镜，然而这些探针在 ROS 后很容易扩散 这会导致 ROS 定位测定不准确和信噪比差。 因此，需要创建能够永久记录响应的探测器。 我们通过荧光成像来对抗基于活性的细胞捕获荧光。 探针可用作进一步了解 ROS 介导的细胞间和细胞内的平台。 我们提出了三个具体目标来检验这一假设。 荧光团与基于活动的触发器和近端氟甲基基团关在一起，作为 ROS 感应的醌方法的潜在等效物将允许 ROS 响应解笼锁。 其次，我们将探针应用于邻近的生物分子。 我们还将绘制多个模型活细胞系来监测 ROS 通量。 该系统使用小胶质细胞-神经元共培养作为生物模型，由 ROS 介导。 我们探测跨细胞氧化还原信号传导，因为小胶质细胞可以在存在的情况下选择性激活 第三个目标是开发一种神经元。 荧光聚合物放大策略以增加串联信号的信噪比 基于活动的传感/标记探针，将主要在 R00 阶段进行。 分子聚合物引发剂将以与前面描述的类似的方式被笼住 荧光探针检测和生物分子标记后，将进行聚合。 从生物分子表面产生荧光聚合物，从而实现信号放大 该策略将被应用到上述活细胞系中。 研究符合申请人的目标，即建立一个利用高分子化学来 探索生物系统中的基本问题。