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

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Modified Project Summary/Abstract Section Elevated levels of reactive oxygen species (ROS) are strongly 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, the capabilities of many ROS sensors are currently still limited by small signal amplitudes, slow kinetics, low sensitivity, in vivo incompatibility, and restraints in cellular and subcellular targeting. Thus, monitoring ROS of oxidative stress in real-time 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 high-throughput screening of large variant libraries in an innovative approach to engineer novel ROS sensors. We expect that significantly increasing signal amplitudes, ROS sensitivity, and insensitivity to hypoxic conditions will enable us to monitor oxidative stress in a wide range of disease models. Furthermore, we will validate new sensors in stem-cell derived models for neurodegeneration and cardiomyopathies with subcellular precision. Our objective is to further maximize ROS sensor function for advanced monitoring of oxidative stress in disease models in response to acute and chronic stressors. In the first aim, we will broaden the color spectrum of this class of sensors by fusing green, yellow, and red fluorescent proteins to a ROS sensitive protein domain . Furthermore, we will create sensors that are more photostable and insensitive to varying oxygen levels compared to fluorescent proteins. In the second aim, we will use a novel engineering platform for fluorescent sensors that allows us 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 the monitoring of oxidative stress as an indicator for Alzheimer’s disease, ischemia and reperfusion 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 a large number of severe diseases. Recent progress in fluorescent microscopy allows us to utilize specific probes to monitoring physiological processes with increasing precision. The engineering of improved ROS sensors will significantly expand the utility of those methods for the analysis of cell signaling and disease progression. Our project is innovative because the proposed approach will provide the fastest throughput for the design of 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.