MacrocyclicPeptide-Based Dual-Sensor Platform forLinkage-Specific Visualization of Ubiquitin Chain Assembling in LiveCells

Intracellular monitoring of protein ubiquitination and differentiating polyubiquitin chain topology are crucial for understanding life processes and drug discovery, which is challenged by the high complexity of the ubiquitination process and a lack of molecular tools. Herein, a synthetic dual-sensor platform specific for K48-linked ubiquitin oligomers was tailored for <i>in situ</i> visualization of polyubiquitin chain assembling in live biosystems. This is achieved using macrocyclic peptides as recognition motifs and a tetraphenylethylene derivative as an activatable reporter. The efficient cell penetration, tight binding, and protection of polyubiquitin delivered a “freeze-and-image” approach, allowing fluorescent readout of polyubiquitin linkage type and chain elongation without perturbing the physiological environment. Motivated by these unique features, mapping of K48-ubiquitination dynamics during protein degradation was facilely achieved. Rapid, sensitive, and intracellular assessment of the mechanism of action and potency dependence for proteolysis-targeting chimeras (PROTACs) was demonstrated, presenting the sensors as promising molecular tools for PROTAC drug development.