Modular Peroxidase-Based Reporters for Detecting Protease Activity and Protein Interactions with Temporal Gating

Enzymatic reporters have been widely applied to study various biological processes because they can amplify signal through enzymatic reactions and provide good sensitivity. However, there is still a need for modular motifs for designing a series of enzymatic reporters. Here, we report a modular peroxidase-based motif, named CLAPon, that features acid-base coil-caged enhanced ascorbate peroxidase (APEX). We demonstrate the modularity of CLAPon by designing a series of reporters for detecting protease activity and protein-protein interactions (PPIs). CLAPon for protease activity showed a 390-fold fluorescent signal increase upon tobacco etch virus protease cleavage. CLAPon for PPI detection (PPI-CLAPon) has two variants, PPI-CLAPon1.0 and 1.1. PPI-CLAPon1.0 showed a signal-to-noise ratio (SNR) of up to 107 for high-affinity PPI pairs and enabled imaging with sub-cellular spatial resolution. However, the more sensitive PPI-CLAPon1.1 is required for detecting low-affinity PPI pairs. PPI-CLAPon1.0 was further engineered to a reporter with light-dependent temporal gating, called LiPPI-CLAPon1.0, which can detect a 3-min calcium-dependent PPI with an SNR of 17. LiPPI-CLAPon enables PPI detection within a specific time window with rapid APEX activation and diverse readout. Lastly, PPI-CLAPon1.0 was designed to have chemical gating, providing more versatility to complement the LiPPI-CLAPon. These CLAPon-based reporter designs can be broadly applied to study various signaling processes that involve protease activity and PPIs and provide a versatile platform to design various genetically encoded reporters.