Development of Activity-Based Reporter Gene Technology for Imaging of Protease Activity with an Exquisite Specificity in a Single Live Cell

Imaging of an active protease with an exquisite specificity in the presence of highly homologous proteins within a living cell is a very challenging task. Herein, we disclose a new method called “Activity-based Reporter Gene Technology” (AbRGT). This method provides an opportunity to study the function of “active protease” with an unprecedented specificity. As a proof-of-concept, we have applied this method to study the function of individual caspase protease in both intrinsic and extrinsic apoptosis signaling pathways. The versatility of this method is demonstrated by studying the function of both the initiator and effector caspases, independently. The modular fashion of this technology provides the opportunity to noninvasively image the function of cathepsin-B in a caspase-dependent cell death pathway. As a potential application, this method is used as a tool to screen compounds that are potent inhibitors of caspases and cathepsin-B proteases. The fact that this method can be readily applied to any protease of interest opens up huge opportunities for this technology in the area of target validation, high-throughput screening, in vivo imaging, diagnostics, and therapeutic intervention.