Structure-guided and high-throughput engineering of genetically encoded sensors for reactive oxygen species

项目摘要/摘要 活性氧(ROS)水平升高与严重的病理条件有关，导致 心肌病和神经退行性变。今天，我们可以利用荧光探针来检测动态变化 细胞生理学和病理生理学中的ROS水平。然而，许多ROS传感器的能力仍然有限 由于信号幅度小，动力学慢，灵敏度低，体内不相容，以及细胞和亚细胞 有针对性的限制。因此，在细胞和动物行为中实时监测ROS仍然是非常有限的。我们的 这项提案的中心目标是解决目前ROS蛋白质传感器的局限性。我们将把结构化的- 引导蛋白质设计和功能高通量筛选创新的大型变异库 设计新型ROS传感器的方法。我们预计，显著增加的信号幅度，动力学 和敏感性，这将使我们能够第一次在行为正常的小鼠的大脑中监测ROS信号。 此外，我们将用亚细胞验证神经退行性变和心肌病模型中的传感器。 人类干细胞来源细胞系的精确度。在第一个目标中，我们将使用蛋白质结构来引导靶向 突变以增加ROS敏感性和传感器和报告结构域之间的变构偶联。在 第二个目标，我们将使用一个新的荧光传感器工程平台来筛选大型文库 随机变种。快速、迭代的过程有可能显著加快优化 目标1中建立了传感器框架。在第三个目标中，我们将在几个实际应用中验证我们的传感器 接收即时反馈以进一步完善传感器功能的场景。这包括监控ROS 作为小鼠行为的第二信使，监测氧化应激作为小鼠病理生理指标 干细胞来源的神经元和心肌细胞。这一建议意义重大，因为氧化应激很常见。 可以影响到每一个器官和细胞类型，导致许多严重的疾病。荧光材料的最新研究进展 显微镜使我们能够利用特定的探针来更精确地监测生理过程。我们的 项目具有创新性，因为所建议的方法将提供最快的吞吐量 高效的ROS感受器蛋白质。此外，改进后的传感器将能够将疾病因果联系起来 氧化应激的急性和慢性应激源的表型在时间和空间上显著增加 决议。 在此，我们请求从CellSorter创新公司购买微流控阀门控制器， 在AIM 2的高通量筛选过程中，匈牙利将更有效地从PDMS微阵列中挑选细胞。 将该仪器集成到我们现有的管道中将进一步增加产量，并将成功率提高到 检索高度优化的ROS传感器，同时减少时间和资源投入。

PROJECT SUMMARY / ABSTRACT Elevated levels of reactive oxygen species (ROS) are linked to severe pathological conditions causing cardiomyopathies and neurodegeneration. Today we can utilize fluorescent probes to detect dynamic changes in ROS levels in cell physiology and pathophysiology. However, many ROS sensors’ capabilities are still limited by small signal amplitudes, slow kinetics, low sensitivity, in vivo incompatibility, and cellular and subcellular targeting restraints. Thus, monitoring ROS in real-time in cells and behaving animals is still very restricted. Our central goal in this proposal is to resolve current limitations in ROS protein sensors. We will combine structured- guided protein design and functional high-throughput screening of large variant libraries in an innovative approach to engineer novel ROS sensors. We expect that significantly increasing signal amplitudes, kinetics and sensitivity, will enable us to monitor ROS signaling for the first time in the brain of behaving mice. Furthermore, we will validate sensors in models for neurodegeneration and cardiomyopathies with subcellular precision in human stem-cell-derived cell lines. In the first aim, we will use protein structures to guide targeted mutations to increase ROS sensitivity and allosteric coupling between the sensor and reporter domain. In the second aim, we will use a novel engineering platform for fluorescent sensors to screen large libraries of randomized variants. The fast, iterative process has the potential to significantly accelerate the optimization of sensor frameworks established in Aim 1. In the third aim, we will validate our sensors in several realistic use scenarios to receive immediate feedback for further refinement of sensor function. This includes monitoring ROS as second messengers in behaving mice and monitoring oxidative stress as an indicator for pathophysiology in stem-cell-derived neurons and cardiomyocytes. This proposal is significant because oxidative stress is common and can affect every organ and cell type resulting in many severe diseases. Recent progress in fluorescent microscopy allows us to utilize specific probes to monitor physiological processes with increasing precision. Our project is innovative because the proposed approach will provide the fastest throughput for designing highly efficient ROS sensor proteins. Furthermore, the improved sensors will be able to causally link disease phenotypes to acute and chronic stressors of oxidative stress with significantly increased temporal and spatial resolution. Here we request the purchase of a microfluidic valve control from CellSorter Company for Innovations, Hungary to more efficiently pick cells from PDMS microarrays during high-throughput screening in Aim 2. Integration of the instrument into our existing pipeline will further increase throughput, and the success rate to retrieve highly optimized ROS sensors while also reducing time and resource commitments.