Suppressor tRNAs, notable for their capability of reading through the stop codon while maintaining normal peptide synthesis, are promising in treating diseases caused by premature termination codons (PTC). However, the lack of effective engineering methods for suppressor tRNAs has curtailed their application potential. Here, we introduce a directed evolution technology that employs phage-assisted continuous evolution (PACE), combined with gradient biosensors featuring various PTCs in the M13 gene III. Utilizing this novel methodology, we have successfully evolved tRNATrp (UGG) reading through the UGA stop codon in Escherichia coli. Massively parallel sequencing revealed that these mutations predominantly occurred in the anticodon loop. Finally, two suppressor tRNATrp (UGA) mutants exhibited over fivefold increases in readthrough efficiency.
机构:
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
Howard Hughes Medical Institute, Harvard University, Cambridge, MADepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
Carlson J.C.
Badran A.H.
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Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
Howard Hughes Medical Institute, Harvard University, Cambridge, MADepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
Badran A.H.
Guggiana-Nilo D.A.
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Harvard Medical School, Boston, MADepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
Guggiana-Nilo D.A.
Liu D.R.
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Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
Howard Hughes Medical Institute, Harvard University, Cambridge, MADepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA
机构:
Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USAHarvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Carlson, Jacob C.
Badran, Ahmed H.
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Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USAHarvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Badran, Ahmed H.
Guggiana-Nilo, Drago A.
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Harvard Univ, Sch Med, Boston, MA USAHarvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Guggiana-Nilo, Drago A.
Liu, David R.
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Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USAHarvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
机构:
Broad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA
Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USABroad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA
Morrison, Mary S.
Wang, Tina
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Broad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA
Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USABroad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA
Wang, Tina
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Raguram, Aditya
Hemez, Colin
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Broad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA
Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USABroad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA
Hemez, Colin
Liu, David R.
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机构:
Broad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA
Harvard Univ, Dept Chem & Chem Biol, Cambridge, MA 02138 USA
Harvard Univ, Howard Hughes Med Inst, Cambridge, MA 02138 USABroad Inst Harvard & MIT, Merkin Inst Transformat Technol Hlth Care, Cambridge, MA 02142 USA