In-brain construction of receptor-based protease sensors by coupling ligand-directed chemistry and click chemistry

The chemical modification of natural proteins in living systems is highly desirable toward the cutting-edge research in chemistry-biology interface. Recent advances in bioorthogonal protein modification have enabled the production of chemically functional proteins in cultured cell systems. However, few methods are applicable in vivo because of the complexity of the three-dimensional constructs of living systems with diverse, heterogeneous cell populations and flow systems filled with tissue fluids. Here, we report a genetic engineering-free method to modify receptor proteins with various probes in the living mouse brain by combining in-brain ligand-directed chemistry with bioorthogonal click chemistry, and propose a chemical guideline for the reaction design. The rapid and selective tethering of a set of fluorescent peptides to AMPA-type glutamate receptors (AMPARs) allowed the construction of receptor-based fluorescent sensors. These probes enabled mapping of the activity of matrix metalloproteinase-9 proximal to AMPARs in the living brain to be realized with high spatial resolution. Our strategy provides new opportunities for the precise analysis of particular in vivo microenvironments that has not been able to be addressed by conventional methods. Such analysis should contribute to the understanding of the molecular basis for complicated in vivo events, such as the regulation of neuroplasticity, the most important challenge in neuroscience.