SHRIMP: Genetically Encoded mScarlet-derived Red Fluorescent Hydrogen Peroxide Sensor with High Brightness and Minimal Photoactivation

Abstract

Red fluorescent protein (RFP) derived indicators are popular due to advantages such as increased imaging depth and reduced autofluorescence and cytotoxicity. However, most RFP-based indicators have low brightness and are susceptible to blue-light-induced photoactivation. In this study, we aimed to overcome the limitations of existing red fluorescent indicators. We utilized mScarlet-I, a highly bright and robust monomeric RFP, to develop a circularly permuted variant called cpmScarlet. We further engineered cpmScarlet into a novel red fluorescent indicator specifically for hydrogen peroxide (H2O2), a crucial reactive oxygen species (ROS) involved in redox signaling and oxidative stress. The resultant indicator, SHRIMP (mScarlet-derived H2O2 Redox Indicator with Minimal Photoactivation), exhibited excitation and emission peaks at [~]570 and 595 nm, respectively, and demonstrated a maximum five-fold fluorescence turn-off response to H2O2. Importantly, SHRIMP was not susceptible to blue-light-induced photoactivation and showed high brightness both in its purified protein form and when expressed in mammalian cells. We successfully employed SHRIMP to visualize H2O2 dynamics in mammalian cells with exogenously added H2O2 and in activated macrophages. Additionally, we demonstrated its utility for multiparameter imaging by co-expressing SHRIMP with GCaMP6m, a green fluorescent calcium indicator, enabling simultaneous monitoring of H2O2 and calcium dynamics in mammalian cells in response to thapsigargin (TG) and epidermal growth factor (EGF) stimulation. Furthermore, we expressed SHRIMP in isolated primary mouse islet tissue, and SHRIMP exhibited excellent brightness and capability for effective detection of H2O2 production during streptozotocin (STZ)-induced {beta}-cell damage. This study successfully transformed mScarlet-I, a bright and robust monomeric RFP, into a circularly permuted variant (cpmScarlet) and developed the first cpmScarlet-based genetically encoded fluorescent indicator called SHRIMP. SHRIMP exhibits high brightness and insensitivity to photoactivation and is a valuable tool for real-time monitoring of H2O2 dynamics in various biological systems. Further research may yield an expanded family of cpmScarlet-based red fluorescent indicators with enhanced photophysical properties.