Site-specific dual encoding and labeling of proteins via genetic code expansion

The ability to selectively modify proteins at two or more defined locations opens new avenues for manipu-lating, engineering, and studying living systems. As a chemical biology tool for the site-specific encoding of non-canonical amino acids into proteins in vivo , genetic code expansion (GCE) represents a powerful tool to achieve such modifications with minimal disruption to structure and function through a two-step "dual encoding and labeling"(DEAL) process. In this review, we summarize the state of the field of DEAL us-ing GCE. In doing so, we describe the basic principles of GCE-based DEAL, catalog compatible encoding systems and reactions, explore demonstrated and potential applications, highlight emerging paradigms in DEAL methodologies, and propose novel solutions to current limitations.