Genetic incorporation of a BCN-bearing amino acid for cell-surface protein labeling via tetrazine ligation.

Tetrazine ligation is a highly attractive bioorthogonal reaction owing to its exceptional selectivity, rapid kinetics, and operationally simple, catalyst-free conditions. Among tetrazine-reactive partners, bicyclo[6.1.0]nonyne (BCN) offers a favorable balance of reagent stability and reactivity. A BCN-bearing non-canonical amino acid (ncAA) can be installed site-specifically into proteins expressed in living prokaryotic or eukaryotic cells through genetic code expansion. In this chapter, we describe detailed procedures to incorporate bicyclo[6.1.0]nonyne-lysine (BCNK) and apply tetrazine ligation to fluorescently label proteins on the surface of live mammalian cells. The workflow is readily adaptable to other tetrazine-reactive amino acids, labeling of cytosolic proteins, and bioorthogonal tethering for functional regulation and other applications.