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Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem ofM. mazeipyrrolysyl tRNA (tRNA^Pyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.

Heterologous tRNAs used for noncanonical amino acid (ncAA) mutagenesis in mammalian cells typically show poor activity. We recently introduced a virus‐assisted directed evolution strategy (VADER) that can enrich improved tRNA mutants from naïve libraries in mammalian cells. However, VADER was limited to processing only a few thousand mutants; the inability to screen a larger sequence space precluded the identification of highly active variants with distal synergistic mutations. Here, we report VADER2.0, which can process significantly larger mutant libraries. It also employs a novel library design, which maintains base‐pairing between distant residues in the stem regions, allowing us to pack a higher density of functional mutants within a fixed sequence space. VADER2.0 enabled simultaneous engineering of the entire acceptor stem ofM. mazeipyrrolysyl tRNA (tRNA^Pyl), leading to a remarkably improved variant, which facilitates more efficient incorporation of a wider range of ncAAs, and enables facile development of viral vectors and stable cell‐lines for ncAA mutagenesis.

We have developed a novel visible‐light‐catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site‐specifically installed 5‐hydroxytryptophan residue (5HTP) on full‐length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light‐emitting diodes (455/650 nm) for rapid site‐specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen‐dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain‐promoted azide‐alkyne click reaction, enables site‐specific dual‐labeling of a target protein.

We have developed a novel visible‐light‐catalyzed bioconjugation reaction, PhotoCLIC, that enables chemoselective attachment of diverse aromatic amine reagents onto a site‐specifically installed 5‐hydroxytryptophan residue (5HTP) on full‐length proteins of varied complexity. The reaction uses catalytic amounts of methylene blue and blue/red light‐emitting diodes (455/650 nm) for rapid site‐specific protein bioconjugation. Characterization of the PhotoCLIC product reveals a unique structure formed likely through a singlet oxygen‐dependent modification of 5HTP. PhotoCLIC has a wide substrate scope and its compatibility with strain‐promoted azide‐alkyne click reaction, enables site‐specific dual‐labeling of a target protein.