Abstract 1934 Genetic Code Expansion for Site-Specific Dual Encoding of Fluorophore-Quencher Pairs

The ability to precisely modify proteins at multiple locations in their natural environment represents an unprecedented opportunity for answering biological questions at the molecular and cellular levels. In this work, we introduce a versatile dual incorporation approach, which involves the site-specific incorporation of two distinct noncanonical amino acids with bioorthogonal properties into proteins in vitro. This innovative strategy has demonstrated remarkable efficacy in incorporating acridone (Acd) and tetrazine (Tet) bearing amino acids at TAG and TAA codons in a single protein using mutually orthogonal tRNA/synthetase sets.