Development and Applications of Bioorthogonal Chemistry: Equipment Supplement ABSTRACT We have a long-standing interest in developing reactivity-based tools to address significant biological problems that are difficult to solve using conventional techniques. In our MIRA application, we plan to continue our studies of orthogonal chemical reactivity in the following three projects. In Project 1, we will design hydrazonyl sultone (HS)-based fluorescence turn-on reagents for constructing FRET-based biosensors to probe class B GPCR activation and signal dynamics in live cells. In Project 2, we will design β-lactam-encoded enzyme traps for covalent capture and subsequent identification of protein lysine methyltransferase substrates in living cells. In Project 3, we will develop covalent nanobody-based immunoPET radiotracers with enhanced performance. The HS-mediated, binding-accelerated ligation reaction will be exploited to achieve greater PET imaging sensitivity without undesired nephrotoxicity. We expect these studies will offer new capabilities of covalent chemistry in biomedical research. This Equipment Supplement requests the acquisition of a Sartorius OCTET-R2 Biolayer Interferometry (BLI) System. This equipment will enable us to rigorously characterize covalent binding of protein substrates toward the designed enzyme traps described in Project 2, and optimize the nanobody-based reaction partners for the HS-based immunoPET radiotracers described in Project 3.