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 manipulating, 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 using 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.