Development and Characterization of Next-Generation Genetically Encodable Fluorescent Biosensors of RSK, S6K, and ROCK Activity

Oluseyi Ochima, Applied Chemistry, College of Science and Technology, North Carolina Agricultural and Technical State University
Henry Uchenna, Applied Chemistry, College of Science and Technology, North Carolina Agricultural and Technical State University
Stephanie Goodrich, Applied Chemistry, College of Science and Technology, North Carolina Agricultural and Technical State University
Debasish Kuila, Applied Chemistry, College of Science and Technology, North Carolina Agricultural and Technical State University

Description

Human diseases like cancer can be attributed to the dysregulation of interrelated signaling pathways. Understanding these processes is crucial for predicting, diagnosing, and treating such diseases. Molecular imaging techniques, such as genetically encodable fluorescence resonance energy transfer (FRET)-based biosensors have been developed to visualize dynamic signaling processes in living cells and organoids. However, they have a low dynamic range, allowing subtle regulatory processes to be missed. Therefore, we developed a series of kinase activity reporters (KARs) (ExRai-RSKAR, ExRai-S6KAR, and ExRai-ROCKAR) with enhanced sensitivity for these kinases based on the recently developed excitation ratiometric indicator (ExRai) architecture, based on the cAMP-dependent protein kinase (PKA) activity reporter, ExRai- AKAR2. These ExRai-based KARs showed enhanced dynamic ranges compared to their FRET counterparts, and they represent valuable tools for monitoring real-time changes in the activity profiles of RSK, ROCK, and S6K with high spatiotemporal resolution in cells and organoids. This approach may help identify biomarkers for associated diseases and examine the responses of these kinases to various pathological, pharmacological, and toxicological agents, improving our understanding of disease mechanisms and facilitating the development of specific therapies.