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

点击翻译按钮获取中文摘要

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.