Multiplex suppression of four quadruplet codons via tRNA directed evolution

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.

Measuring the tolerance of the genetic code to altered codon size

“…Many qtRNAs were quite inefficient in translation: the presence of a single quadruplet codon in an mRNA transcript can reduce total protein yield to less than 3% relative to an all-triplet mRNA ( Figure 2 ). Mutations at the anticodon loop sides of the qtRNAs have been observed to improve translation efficiency for TAGA-qtRNAs ( DeBenedictis et al, 2021 ; DeBenedictis et al, 2022 ; Niu et al, 2013 ). Triplet tRNAs are known to exhibit patterns that relate the bases in the anticodon loop sides to the bases in the anticodon itself ( Yarus, 1982 ), and similarly benefit from anticodon loop side mutations after anticodon replacement ( Kleina et al, 1990 ; Raftery and Yarus, 1987 ; Cervettini et al, 2020 ).…”

“…Triplet tRNAs are known to exhibit patterns that relate the bases in the anticodon loop sides to the bases in the anticodon itself ( Yarus, 1982 ), and similarly benefit from anticodon loop side mutations after anticodon replacement ( Kleina et al, 1990 ; Raftery and Yarus, 1987 ; Cervettini et al, 2020 ). In some cases, mutations in this area can alter qtRNA charging; in others they improve quadruplet translation efficiency without altering the qtRNA’s interaction with the cognate AARS ( DeBenedictis et al, 2021 ). We hypothesized that qtRNAs in general require mutations at bases 32, 37, and 38 to better accommodate a new codon, and that this requirement may present a key barrier preventing the natural evolution of efficient qtRNAs.…”

“…We began by assessing results for the well-studied qtRNA Ser TAGA , which is known to have an improved variant, qtRNA Ser TAGA -32A-38C, that exhibits improved quadruplet codon translation but unaltered, selective aminoacylation with serine ( DeBenedictis et al, 2021 ; Figure 4C ). Of the 64 possible combinations of DNA bases at positions 32, 37, and 38, the single library member A32 A37 C38 is enriched four log fold above all other variants ( Figure 4D ).…”

“…Many qtRNAs were quite inefficient in translation: the presence of a single quadruplet codon in an mRNA transcript can reduce total protein yield to less than 3% relative to an all-triplet mRNA (Figure 2). Mutations at the anticodon loop sides of the qtRNAs have been observed to improve translation efficiency for TAGA-qtRNAs 9,13,16 . Triplet tRNAs are known to exhibit patterns that relate the bases in the anticodon loop sides to the bases in the anticodon itself 17…”

“…An expanded all-quadruplet genetic code would offer 256 total codons ( de la Torre and Chin, 2021 ), including hundreds of free codons that could be assigned to non-canonical amino acids (ncAAs), valuable chemical additions to the genetic code that enable improved protein therapeutics ( Hutchins et al, 2011 ). Indeed, the efficiency of quadruplet translation can be so high that as many as four unique quadruplets can be translated within one transcript to incorporate natural ( DeBenedictis et al, 2021 ) or non-canonical ( Dunkelmann et al, 2021 ) amino acids site-specifically. These inspiring results raise the question: is it possible to create an all-quadruplet genetic code?…”