Multiplex suppression of four quadruplet codons via tRNA directed evolution.

Autor: DeBenedictis EA; The Broad Institute of MIT & Harvard, Cambridge, MA, USA.; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA., Carver GD; The Broad Institute of MIT & Harvard, Cambridge, MA, USA., Chung CZ; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA., Söll D; Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.; Department of Chemistry, Yale University, New Haven, CT, USA., Badran AH; The Broad Institute of MIT & Harvard, Cambridge, MA, USA. ahbadran@scripps.edu.; Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA. ahbadran@scripps.edu.
Jazyk: angličtina
Zdroj: Nature communications [Nat Commun] 2021 Sep 29; Vol. 12 (1), pp. 5706. Date of Electronic Publication: 2021 Sep 29.
DOI: 10.1038/s41467-021-25948-y
Abstrakt: Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.
(© 2021. The Author(s).)
Databáze: MEDLINE
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