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

基本信息

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
项目类别：
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

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

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项目摘要

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.

项目摘要 /摘要活性氧（ROS）的水平升高与导致严重的病理状况有关心肌病和神经变性。今天，我们可以利用荧光问题来检测动态变化在细胞生理和病理生理学中ROS水平中。但是，许多ROS传感器的功能仍然有限通过小信号放大器，缓慢的动力学，低灵敏度，体内不兼容，细胞和亚细胞定位约束。那是在细胞和行为动物中实时监测ROS仍然非常限制。我们的该提案中的中心目标是解决ROS蛋白传感器中的当前局限性。我们将结合结构化 - 在创新中对大型变体库的指导蛋白质设计和功能性高通量筛选工程师小说ROS传感器的方法。我们预计会显着增加信号放大器，动力学敏感性将使我们能够在行为小鼠的大脑中首次监测ROS信号。此外，我们将验证具有亚细胞的神经变性和心肌病模型的传感器人类干细胞衍生的细胞系中的精度。在第一个目标中，我们将使用蛋白质结构来指导目标突变以提高传感器和报道域之间的ROS灵敏度和变构耦合。在第二个目的，我们将使用一个新颖的工程平台用于荧光传感器来筛选大型库随机变体。快速的迭代过程有可能显着加速优化在AIM 1中建立的传感器框架。在第三个目标中，我们将在几种现实使用中验证我们的传感器即时反馈以进一步完善传感器功能的情况。这包括监视ROS 作为在行为小鼠和监测氧化应激中的第二个使者，作为病理生理学的指标干细胞衍生的神经元和心肌细胞。该建议很重要，因为氧化应激很常见并可能影响导致许多严重疾病的每种器官和细胞类型。荧光的最新进展显微镜使我们能够利用特定的问题以提高精度监视物理过程。我们的项目具有创新性，因为拟议的方法将为高度设计提供最快的吞吐量有效的ROS传感器蛋白。此外，改进的传感器将能够因果关系连接疾病氧化应激的急性和慢性应激的表型，暂时和空间显着增加解决。在这里，我们要求从Cellsorter Company购买创新的微流体阀门控制，匈牙利在AIM 2中的高通量筛选期间从PDMS微阵列中挑选PDMS微阵列的细胞。将仪器集成到我们现有的管道中将进一步增加吞吐量，并将成功率提高到检索高度优化的ROS传感器，同时还减少了时间和资源承诺。