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

活性氧基因编码传感器的结构引导和高通量工程

• 批准号: 10797426
• 负责人: Andre Berndt
• 金额: $ 2.35万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797426
• 关键词: Acceleration; Acute; Affect; Animals; Brain; Cardiac Myocytes; Cardiomyopathies; Cell Line; Cell physiology; Cells; Chronic; Coupling; Disease; Drug Screening; Engineering; Feedback; Fluorescent Probes; Functional disorder; Genetic Engineering; Goals; Hungary; Kinetics; Libraries; Link; Location; Microfluidics; Microscopy; Modeling; Monitor; Mus; Mutation; Nerve Degeneration; Neurons; Organ; Oxidative Stress; Pathologic; Physiologic Monitoring; Physiological Processes; Process; Protein Engineering; Proteins; Randomized; Reactive Oxygen Species; Reporter; Research Personnel; Resolution; Resources; Second Messenger Systems; Signal Transduction; Speed; Stress; Structure; Testing; Time; Variant; cell type; cellular targeting; design; disease phenotype; high throughput screening; human stem cells; improved; in vivo; innovation; instrument; novel; novel therapeutic intervention; protein structure; restraint; sensor; stem cells; stressor; subcellular targeting; success

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.

项目总结/摘要 活性氧（ROS）水平升高与严重的病理状况有关， 心肌病和神经变性。今天，我们可以利用荧光探针来检测动态变化 在细胞生理学和病理生理学中的活性氧水平。然而，许多ROS传感器的能力仍然有限 通过小信号幅度、慢动力学、低灵敏度、体内不相容性、以及细胞和亚细胞 瞄准限制。因此，实时监测细胞和行为动物中的ROS仍然非常有限。我们 该提案的中心目标是解决ROS蛋白传感器的现有局限性。我们将联合收割机- 指导蛋白质设计和功能性高通量筛选大型变异库， 设计新型ROS传感器的方法我们预期显著增加的信号幅度、动力学 和灵敏度，将使我们能够监测活性氧信号的第一次在大脑中的行为小鼠。 此外，我们将在神经变性和心肌病模型中验证传感器， 人类干细胞衍生细胞系的精确度。在第一个目标中，我们将使用蛋白质结构来引导靶向的 突变以增加ROS敏感性和传感器与报告结构域之间的变构偶联。在 第二个目标，我们将使用一种新的荧光传感器工程平台来筛选大型的 随机变量。快速的迭代过程有可能显著加速 Aim 1中建立的传感器框架。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 场景接收即时反馈，以进一步完善传感器功能。这包括监测ROS 作为第二信使在行为小鼠和监测氧化应激作为指标的病理生理学， 干细胞衍生的神经元和心肌细胞。这一建议意义重大，因为氧化应激是常见的 并且可以影响每个器官和细胞类型，导致许多严重的疾病。荧光染料的最新进展 显微镜使我们能够利用特定的探针以越来越高的精度监测生理过程。我们 项目是创新的，因为所提出的方法将提供最快的吞吐量设计高度 高效ROS传感器蛋白。此外，改进的传感器将能够将疾病与疾病之间的因果关系联系起来， 急性和慢性氧化应激应激表型与显着增加的时间和空间 分辨率 在这里，我们要求从CellSorter公司购买微流体阀控制创新， 匈牙利在Aim 2的高通量筛选过程中更有效地从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吞吐量， 检索高度优化的ROS传感器，同时还减少了时间和资源的承诺。