Abstract

Expanding the genetic code has revolutionized our ability to study and manipulate biological systems through site-specific incorporation of noncanonical amino acids (ncAAs). However, current methods are primarily limited to single-type ncAA incorporation in mammalian cells owing to translation inefficiency. Here we introduce a multi-type rare codon recoding strategy that addresses this limitation. By systematically evaluating and repurposing rare codons, alongside engineering mutually orthogonal aminoacyl-tRNA synthetase/tRNA pairs, we achieve the expression of proteins containing two or three distinct ncAAs at site-specific positions with recoding rates of up to 90% at wild-type protein expression levels in mammalian cells. This approach facilitates a broad range of applications, including dual bioorthogonal labelling and sequential protein activation. We further demonstrate the utility of this strategy by incorporating up to five distinct ncAAs into a single protein, revealing a redefinable nature of the genetic code and opening unprecedented avenues for future applications in biomedicine and synthetic biology.