Effect of neighboring nucleotide sequences on suppression efficiency in amber mutants of T4 phage lysozyme

Akaboshi, Eiko; Inouye, Masayori; Tsugita, Akira

doi:10.1007/bf00275955

Cited by 29 publications

(1 citation statement)

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“…Notably, codon−anticodon pairs derived from lacZ selections did not always yield measurable luminescence activity upon supplementary validation in the LuxAB reporter system. This may suggest that at least some selection-derived qtRNAs can suffer from context-dependent effects [52][53][54][55] . Nonetheless, many qtRNAs showed robust decoding efficiencies when assayed in the LuxAB reporter assay, reaching up to η = 6% (Fig.…”

Section: Resultsmentioning

confidence: 99%

Multiplex suppression of four quadruplet codons via tRNA directed evolution

et al. 2021

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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.

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Section: Resultsmentioning

confidence: 99%