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1.
ACS Synth Biol ; 13(7): 2141-2149, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38904157

RESUMO

The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeu could be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.


Assuntos
Aminoácidos , Evolução Molecular Direcionada , Escherichia coli , Mutagênese , Evolução Molecular Direcionada/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Aminoácidos/genética , Aminoácidos/metabolismo , Células HEK293 , Leucina-tRNA Ligase/genética , Leucina-tRNA Ligase/metabolismo
2.
Angew Chem Int Ed Engl ; 63(9): e202316428, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38279536

RESUMO

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 of M. mazei pyrrolysyl tRNA (tRNAPyl ), 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.


Assuntos
Aminoácidos , Aminoacil-tRNA Sintetases , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Escherichia coli/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Mutagênese
3.
Nat Methods ; 20(1): 95-103, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36550276

RESUMO

Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-transfer RNA synthetase-tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often have poor intrinsic activity, presumably due to suboptimal interaction with a non-native translation system. This limitation can be addressed in Escherichia coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which uses a double-sieve selection scheme to facilitate single-step enrichment of active yet orthogonal tRNA mutants from naive libraries. Using VADER we developed improved mutants of Methanosarcina mazei pyrrolysyl-tRNA, as well as a bacterial tyrosyl-tRNA. We also show that the higher activity of the most efficient mutant pyrrolysyl-tRNA is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation apparatus.


Assuntos
Aminoacil-tRNA Sintetases , RNA de Transferência , RNA de Transferência/genética , RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo
4.
Biochemistry ; 60(7): 489-493, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33560840

RESUMO

The ability to engineer the substrate specificity of natural aminoacyl-tRNA synthetase/tRNA pairs facilitates the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. The Methanocaldococcus jannaschii-derived tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair has been engineered to incorporate numerous ncAAs into protein expressed in bacteria. However, it cannot be used in eukaryotic cells due to cross-reactivity with its host counterparts. The Escherichia coli-derived tyrosyl-tRNA synthetase (EcTyrRS)/tRNA pair offers a suitable alternative to this end, but a much smaller subset of ncAAs have been genetically encoded using this pair. Here we report that this discrepancy, at least partly, stems from the structural robustness of EcTyrRS being lower than that of MjTyrRS. We show that the thermostability of engineered TyrRS mutants is generally significantly lower than those of their wild-type counterparts. Derived from a thermophilic archaeon, MjTyrRS is a remarkably sturdy protein and tolerates extensive active site engineering without a catastrophic loss of stability at physiological temperature. In contrast, EcTyrRS exhibits significantly lower thermostability, rendering some of its engineered mutants insufficiently stable at physiological temperature. Our observations identify the structural robustness of an aaRS as an important factor that significantly influences how extensively it can be engineered. To overcome this limitation, we have further developed chimeras between EcTyrRS and its homologue from a thermophilic bacterium, which offer an optimal balance between thermostability and activity. We show that the chimeric bacterial TyrRSs show enhanced tolerance for destabilizing active site mutations, providing a potentially more engineerable platform for genetic code expansion.


Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Engenharia de Proteínas/métodos , Aminoácidos/genética , Aminoacil-tRNA Sintetases/genética , Domínio Catalítico/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Código Genético/genética , RNA de Transferência/metabolismo , Especificidade por Substrato/genética , Tirosina-tRNA Ligase/química , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo
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