Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase–tRNA pairs

Abstract

A central challenge in expanding the genetic code of cells to incorporate non-canonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)-tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach -named tRNA Extension (tREX) -to determine the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs. We discover five orthogonal pairs, including three highly active amber suppressors, and evolve new amino acid substrate specificities for two pairs. Finally, we use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expands the number of orthogonal pairs available for genetic code expansion and provides a pipeline for the discovery of additional orthogonal pairs and a foundation for encoding the cellular synthesis of non-canonical biopolymers.Genetic code expansion enables the cellular synthesis of modified proteins via the cotranslational incorporation of non-canonical amino acids 1, 2 . Orthogonal aaRS-tRNA pairs are crucial to genetic code expansion. These pairs consist of (1) a synthetase that efficiently aminoacylates its cognate tRNA, but minimally aminoacylates endogenous tRNAs in the host organism, and (2) a tRNA that is a substrate for its cognate synthetase but is a poor substrate for endogenous synthetases 3,4 . Derivatives of orthogonal pairs that recognize blank codons (most commonly, the amber stop codon) and selectively use non-canonical amino acids (ncAAs) have been used to site-specifically incorporate numerous ncAAs into proteins 3,4 . Despite many years of effort, a limited number of orthogonal aaRS-tRNA pairs *

Rational Design of Aptamer-Tagged tRNAs

“…It is well known that a large subgroup of α-Proteobacteria has a non-canonical tRNA His lacking the G-1:C73 pair and has a non-canonical HisRS species that recognizes the non-canonical G1:U72 pair and A73 in addition to the GUG anticodon [ 17 , 57 , 58 , 59 , 60 ]. The two α-proteobacterial species are Afifella pfennigii DSM 17,143 ( Ap ) [ 17 ] and Consotaella salsifontis USBA 369 ( Cs ) [ 61 ], because the well-studied α-proteobacterial Caulobacter crescentus HisRS was not active enough in E. coli [ 60 ]. Two kinds of allo-tRNA chassis, S072 and S005, were engineered to have a typical α-proteobacterial tRNA His anticodon arm as their V-arms, to develop H072-αψHis-3 and αH005-αψHis-3/4/5 variants ( Figure 7 A).…”

“…tRNA plays a central role in reprogramming the genetic code system and establishing new genetic codes [ 16 , 17 , 18 ]. In nature, the genetic code may have been expanded to acquire selenocysteine (Sec) and pyrrolysine (Pyl) as the 21st and 22nd amino acid, respectively.…”

“…Interestingly, canonical tRNA Sec has the largest tRNA structure, while tRNA Pyl has the most compact tRNA structure [ 16 ], which may help them escape from non-cognate aminoacyl-tRNA synthetases (aaRSs). The genetic code was expanded in diverse organisms using synthetic biologists by developing various sets of orthogonal tRNA and aaRS pairs that do not cross-react with endogenous tRNAs and aaRSs and other orthogonal tRNA-aaRS pairs [ 17 , 18 , 19 , 20 , 21 , 22 ]. It has been a big challenge to artificially create orthogonal tRNA species with both high tRNA activity and high orthogonality in part due to saturation of recognition elements of tRNA [ 23 ].…”

“…It has been a big challenge to artificially create orthogonal tRNA species with both high tRNA activity and high orthogonality in part due to saturation of recognition elements of tRNA [ 23 ]. One promising approach is “tRNA Extension (tREX)” [ 17 ] by which the tRNA activity and the orthogonality of a few orthogonal systems were significantly enhanced in a rapid and systematic manner towards their simultaneous use in E. coli [ 17 ]. Creation of a new tRNA identity may be achieved by introducing new tertiary structural elements [ 24 , 25 ] rather than by introducing small local modifications to tRNA.…”

An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

“…The plasmid harbors an orthogonal translation system (OTS), as well as a reporter gene for system validation. An OTS, composed of a tRNA and tRNA-synthetase pair, could be considered orthogonal if it does not interact with native translational components 26 . Methanosarcina mazei pyrrolysyl orthogonal translation system 27 (MmPyl OTS) has been previously found to be orthogonal in several organisms, including gram-negative bacteria, and was therefore our choice.…”