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

项目摘要 活性氧簇(ROS)是生命系统中的一类小分子，为生命系统提供重要的 在信号和压力两个方面都有作用。过氧化氢(H_2O_2)、超氧化物(O_2·-)和次氯 除其他外，酸(HOCl)都是传统上被视为 氧化应激和损伤的来源。ROS的异常产生导致了大量的 神经退行性变、癌症和心血管疾病等病理疾病。然而，ROS 也是通过激活多个类别的 蛋白质。这种信号-应力二分法，再加上ROS的小和瞬时性质， 在尝试解码细胞氧化还原的复杂图景时提出了一个挑战 动态平衡。荧光探针经常被用来显示生命系统中的ROS 然而，通过荧光显微镜观察，这些探针在ROS后容易扩散 侦测。这导致了ROS定位的不准确确定和较差的信噪比 回应。因此，需要创建服从永久记录的探测器 ROS通过荧光成像。我们假设基于活性的细胞可捕捉荧光 探针可以作为一个平台来进一步了解ROS介导的细胞内和细胞内 细胞信号。我们提出了三个具体目标来检验这一假设。首先，我们将合成 用基于活性的触发剂和近端的氟甲基作为笼中的荧光团作为 ROS感测中的潜在等价物甲基苯醌。ROS响应取消将允许 用于邻近生物分子的荧光标记。第二，我们将把我们的探测器应用于 用于监测ROS通量的多个活细胞模型。我们还将映射细胞到细胞的通信 以小胶质细胞-神经元共培养为生物学模型的ROS介导。这个系统将允许 美国将探索跨细胞氧化还原信号，因为在存在的情况下，小胶质细胞可以被选择性地激活 从而将过氧化氢分派给附近的神经元。第三个目标涉及开发一种 提高串联系统信噪比响应的荧光聚合物放大策略 以活动为基础的传感/标记探针，将主要在R00阶段进行。小的- 分子聚合物引发剂将以与前面描述的类似的方式被笼化 荧光探头。在ROS传感和生物分子标记后，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 和视觉化。这一策略将被应用到上述活细胞系中。这 研究符合申请者的目标，即建立一个使用聚合物化学来 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.