Protease-Containing Nanobodies for Detecting and Manipulating Intracellular Antigens Using Antiviral Drugs

Tools to induce the formation of protein-protein interactions (PPIs) via small molecules are essential for investigating and engineering biological systems. Here, we introduce a protease-based strategy for controlling the preservation of otherwise self-cleaving nanobodies. By inserting the Hepatitis C Virus NS3 cis-protease into the nanobody scaffold, we showed that the antigen-binding ability of these chimeric nanobodies can be controlled in a dose-dependent manner using NS3 inhibitors. We demonstrated the generalizability of this approach by designing and validating drug-controllable nanobodies targeting mCherry (LaM4), eGFP (LaG16), and the ALFA peptide tag. Additionally, we showed that an NS3-containing version of a nanobody targeting the β2-adrenergic receptor can control endogenous G-protein-mediated signaling activity. Overall, we introduce new chemogenetic components for controlling intracellular PPIs using clinically approved antiviral drugs.