Engineered triply orthogonal pyrrolysyl–tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids

Abstract

Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyltransfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/ Pyl tRNA pairs are the most widely used system for genetic code expansion. Here we reveal that the sequences of ΔNPylRS/ ΔNPyl tRNA pairs (which lack Nterminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyl tRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyl tRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ ΔNPyl tRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/ Pyl tRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.

Advances and Opportunities in Epigenetic Chemical Biology

“…Similarly, another recent study demonstrated that an evolved leucyl aaRS from E. coli is orthogonal to the M. barkeri PylRS in mammalian cells, again allowing the incorporation of two ncAAs [361] . Notably, a recent study has also developed a method for incorporating three ncAAs in E. coli , using engineered triply orthogonal aaRS/tRNA pairs to suppress a nonsense codon and two quadruplet codons [362] . With these advances, the incorporation of more than one ncAA within a single target becomes possible, widely expanding the study of PTM combinations.…”

Site-selective modification strategies in antibody–drug conjugates

“…197 To date, up to three different ncAAs have been incorporated into a single polypeptide chain using multiple orthogonal aaRS/tRNA pairs. 198 However, there is only one example of multiple ncAAs being used for the synthesis of a dual-functional ADC (Fig. 12A).…”

Using Genetic Code Expansion for Protein Biochemical Studies

“…More recently, two separate classes of pyrrolysyl-tRNA synthetase (PylRS) with the N-terminal domain missing and their corresponding tRNAs were identified to be mutually orthogonal to each other and to a Methanosarcina mazei PylRS/SpetRNA Pyl pair in E coli. By co-expressing all three pairs and ribo-Q1 with a GFP reporter, three distinct ncAAs were incorporated by recoding the UAG stop codon and two quadruplet codons (Dunkelmann et al, 2020). The use of quadruplet codons would allow protein termination with a triplet stop codon.…”