Multiplex suppression of four quadruplet codons via tRNA directed evolution

Erika A Debenedictis,Christina Z Chung,Dieter Söll,Gavriela D Carver,Ahmed H Badran

doi:10.1038/s41467-021-25948-y

Erika A Debenedictis, Christina Z Chung + Show 3 more

Open Access

https://doi.org/10.1038/s41467-021-25948-y

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Abstract

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.

Highlights

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
We confirmed that luminescence relies on the presence of a quadruplet-decoding tRNAs (qtRNAs) and correct codon−anticodon interaction (Fig. 1b), and validated it using three previously reported qtRNAs (Fig. 1b, c and Supplementary Table 1)
We unify our nomenclature by referring to qtRNAs as qtRNAscaffoldcodon; e.g., qtRNATyrUAGA is a tyrosine tRNA scaffold containing a 5′-UCUA-3′ anticodon that should decode the cognate UAGA quadruplet codon in an mRNA

Summary

Introduction

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. 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. Genetic code expansion (GCE) has enabled the programmed incorporation of non-canonical amino acids (ncAAs) in proteins in living cells This has been achieved —in nature and in the laboratory—by recoding existing redundant stop or sense codons[1,2,3,4,5,6], increasing codon size[7,8,9,10,11,12], or by adding additional synthetic letters to the genetic language[13,14]. Implementing such a system will require efficient, well-characterized tRNAs capable of translating quadruplet codons into the corresponding canonical amino acids (cAAs), alongside diverse ncAAs for researcher-dictated functions

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