Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs.

Mutually orthogonal aminoacyl transfer RNA synthetase/transfer RNA pairs provide a foundation for encoding non-canonical amino acids into proteins, and encoded non-canonical polymer and macrocycle synthesis. Here we discover quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/pyrrolysyl-tRNA (tRNAPyl) pairs. We discover empirical sequence identity thresholds for mutual orthogonality and use these for agglomerative clustering of PylRS and tRNAPyl sequences; this defines numerous sequence clusters, spanning five classes of PylRS/tRNAPyl pairs (the existing classes +N, A and B, and newly defined classes C and S). Most of the PylRS clusters belong to classes that were unexplored for orthogonal pair generation. By testing pairs from distinct clusters and classes, and pyrrolysyl-tRNAs with unusual structures, we resolve 80% of the pairwise specificities required to make quintuply orthogonal PylRS/tRNAPyl pairs; we control the remaining specificities by engineering and directed evolution. Overall, we create 924 mutually orthogonal PylRS/tRNAPyl pairs, 1,324 triply orthogonal pairs, 128 quadruply orthogonal pairs and 8 quintuply orthogonal pairs. These advances may provide a key foundation for encoded polymer synthesis.

Mechanism-based traps enable protease and hydrolase substrate discovery.

Hydrolase enzymes, including proteases, are encoded by 2-3% of the genes in the human genome and 14% of these enzymes are active drug targets1. However, the activities and substrate specificities of many proteases-especially those embedded in membranes-and other hydrolases remain unknown. Here we report a strategy for creating mechanism-based, light-activated protease and hydrolase substrate traps in complex mixtures and live mammalian cells. The traps capture substrates of hydrolases, which normally use a serine or cysteine nucleophile. Replacing the catalytic nucleophile with genetically encoded 2,3-diaminopropionic acid allows the first step reaction to form an acyl-enzyme intermediate in which a substrate fragment is covalently linked to the enzyme through a stable amide bond2; this enables stringent purification and identification of substrates. We identify new substrates for proteases, including an intramembrane mammalian rhomboid protease RHBDL4 (refs. 3,4). We demonstrate that RHBDL4 can shed luminal fragments of endoplasmic reticulum-resident type I transmembrane proteins to the extracellular space, as well as promoting non-canonical secretion of endogenous soluble endoplasmic reticulum-resident chaperones. We also discover that the putative serine hydrolase retinoblastoma binding protein 9 (ref. 5) is an aminopeptidase with a preference for removing aromatic amino acids in human cells. Our results exemplify a powerful paradigm for identifying the substrates and activities of hydrolase enzymes.

A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design.

Orthogonal (O) ribosome-mediated translation of O-mRNAs enables the incorporation of up to three distinct non-canonical amino acids (ncAAs) into proteins in Escherichia coli (E. coli). However, the general and efficient incorporation of multiple distinct ncAAs by O-ribosomes requires scalable strategies for both creating efficiently and specifically translated O-mRNAs, and the compact expression of multiple O-aminoacyl-tRNA synthetase (O-aaRS)/O-tRNA pairs. We automate the discovery of O-mRNAs that lead to up to 40 times more protein, and are up to 50-fold more orthogonal, than previous O-mRNAs; protein yields from our O-mRNAs match or exceed those from wild-type mRNAs. These advances enable a 33-fold increase in yield for incorporating three distinct ncAAs. We automate the creation of operons for O-tRNA genes, and develop operons for O-aaRS genes. Combining our advances creates a 68-codon, 24-amino-acid genetic code to efficiently incorporate four distinct ncAAs into a single protein in response to four distinct quadruplet codons.

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

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 aminoacyl-transfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/ΔNPyltRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyltRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyltRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ΔNPyltRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/PyltRNA 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.