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

项目摘要 活性氧（ROS）是生命系统中的一个小分子家族， 在信号和压力中的作用。过氧化氢（H2 O2）、超氧化物（O2·-）和次氯酸 酸（HOCl）等都是传统上被视为ROS的例子。 氧化应激和损伤的来源。异常的ROS产生导致了许多 病理学如神经变性、癌症和心血管疾病。然而，ROS 对于通过激活多种类型的代谢酶来维持代谢稳态也是至关重要的。 proteins.这种信号-应激二分法，加上ROS的小而短暂的性质， 当试图解码细胞氧化还原的复杂景观时， 体内平衡荧光探针经常被用来观察生命系统中的活性氧 通过荧光显微镜，然而，这些探针在ROS后易于扩散， 侦测这导致ROS定位的不准确测定和差的信噪比 应答因此，需要创建适合于永久记录的探针， ROS通过荧光成像。我们假设基于活性的细胞可捕获荧光 探针可用作进一步了解ROS介导的细胞间和细胞内 细胞信号我们提出了三个具体目标来检验这一假设。首先，我们将综合 用基于活性的触发剂和近端氟甲基笼住的荧光团， 潜在当量的醌甲基化物的ROS传感。ROS响应性撑开将允许 用于相邻生物分子的荧光标记。第二，我们将把我们的探测器应用于 多个模型活细胞系以监测ROS通量。我们还将绘制细胞间的通讯 使用小胶质细胞-神经元共培养物作为生物模型，由活性氧介导。该系统将允许 我们探测跨细胞氧化还原信号，因为小胶质细胞可以选择性地激活， 从而将H2 O2发送到附近的神经元。第三个目标是发展一个 荧光聚合物扩增策略，以增加串联的信噪比响应 基于活性的传感/标记探针，并将主要在R 00阶段进行。小- 分子聚合物引发剂将以与前述类似的方式被笼化 荧光探针。在ROS感测和生物分子标记之后，将进行聚合 从生物分子表面产生荧光聚合物， 和可视化。该策略将延续到上述活细胞系中。这 研究符合申请人的目标，即建立一个使用聚合物化学的程序， 探索生物系统中的基本问题。

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.