Abstract: The incorporation of non-canonical amino acids (ncAAs) has expanded the chemical diversity and functional repertoire of proteins beyond the 20 canonical amino acids. While stop codon suppression introduces a single orthogonal decoding event, sense codon reassignment actively manipulates endogenous translational competition within the ribosomal decoding center. This strategy enables both proteome-wide stochastic labeling and precise site-specific modification of target proteins. In this review, we highlight recent advances in sense codon recoding strategies and their applications in nascent proteome profiling and precision protein engineering. We first discuss global metabolic labeling using endogenous aminoacyl-tRNA synthetases and the programmable Stochastic Orthogonal Recoding of Translation (SORT) platform. We then examine site-specific approaches, including competitive rare codon recoding in mammalian cells, genome-scale codon compression in Escherichia coli, and the creation of blank coding channels through total genome synthesis. These complementary paradigms have enabled high-resolution spatiotemporal mapping of nascent proteomes in live animals, multiplexed incorporation of up to five distinct ncAAs in a single mammalian protein, and the in vivo biosynthesis of complex sequence-defined polymers. By converting translational competition into an engineerable parameter, sense codon reassignment offers new opportunities to transform our ability to decode complex biological networks and develop next-generation molecular medicines.

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