tRNA shape is an identity element for an archaeal pyrrolysyl-tRNA synthetase from the human gut.

Protein translation is orchestrated through tRNA aminoacylation and ribosomal elongation. Among the highly conserved structure of tRNAs, they have distinguishing features which promote interaction with their cognate aminoacyl tRNA synthetase (aaRS). These key features are referred to as identity elements.

Reprogramming Initiator and Nonsense Codons to Simultaneously Install Three Distinct Noncanonical Amino Acids into Proteins in E. coli.

Multiple noncanonical amino acids can be installed into proteins in E. coli using mutually orthogonal aminoacyl-tRNA synthetase and tRNA pairs. Here we describe a protocol for simultaneously installing three distinct noncanonical amino acids into proteins for site-specific bioconjugation at three sites.

Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression.

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells.

Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2.

Using the regioselective cyanobenzothiazole condensation reaction with an N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the novel severe acute respiratory syndrome virus 2 (SARS-CoV-2) Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and angiotensin-converting enzyme 2 (ACE2), the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2.

Ancestral archaea expanded the genetic code with pyrrolysine.

The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea.

Directed Evolution of Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant.

Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of PylRS (PylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of PylRS have not been reported.

Initiating protein synthesis with noncanonical monomers in vitro and in vivo.

With few exceptions, ribosomal protein synthesis begins with methionine (or its derivative N-formyl-methionine) across all domains of life. The role of methionine as the initiating amino acid is dictated by the unique structure of its cognate tRNA known as tRNA. By mis-acylating tRNA, we and others have shown that protein synthesis can be initiated with a variety of canonical and noncanonical amino acids both in vitro and in vivo.

Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons.

We recently described an orthogonal initiator tRNA (tRNA) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here, we report that a mutant of tRNA (tRNA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing tRNA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and predominantly tyrosine at elongating positions.

An amber obligate active site-directed ligand evolution technique for phage display.

Although noncanonical amino acids (ncAAs) were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages. Here, we report a technique based on superinfection immunity of phages to enrich amber-containing clones, thus avoiding the observed bias that has hindered incorporation of ncAAs into phage libraries. We then take advantage of this technique for development of active site-directed ligand evolution of peptides, where the ncAA serves as an anchor to direct the binding of its peptides to the target's active site.

Hijacking Translation Initiation for Synthetic Biology.

Genetic code expansion (GCE) has revolutionized the field of protein chemistry. Over the past several decades more than 150 different noncanonical amino acids (ncAAs) have been co-translationally installed into proteins within various host organisms. The vast majority of these ncAAs have been incorporated between the start and stop codons within an open reading frame.

Engineering aminoacyl-tRNA synthetases for use in synthetic biology.

Within the broad field of synthetic biology, genetic code expansion (GCE) techniques enable creation of proteins with an expanded set of amino acids. This may be invaluable for applications in therapeutics, bioremediation, and biocatalysis. Central to GCE are aminoacyl-tRNA synthetases (aaRSs) as they link a non-canonical amino acid (ncAA) to their cognate tRNA, allowing ncAA incorporation into proteins on the ribosome.

Initiation of Protein Synthesis with Non-Canonical Amino Acids In Vivo.

By transplanting identity elements into E. coli tRNA , we have engineered an orthogonal initiator tRNA (itRNA ) that is a substrate for Methanocaldococcus jannaschii TyrRS. We demonstrate that itRNA can initiate translation in vivo with aromatic non-canonical amino acids (ncAAs) bearing diverse sidechains.

A Genetically Encoded, Phage-Displayed Cyclic-Peptide Library.

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands for therapeutic targets, phage-displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage-display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded N -acryloyl-lysine (AcrK).

Using Amber and Ochre Nonsense Codons to Code Two Different Noncanonical Amino Acids in One Protein Gene.

In Escherichia coli, conventional amber and ochre stop codons can be separately targeted by engineered amber-suppressing Methanocaldococcus jannaschii tyrosyl-tRNA synthetase-tRNA and ochre-suppressing Methanosarcina maezi pyrrolysyl-tRNA synthetase-tRNA pairs for coding two different noncanonical amino acids in one protein gene. Here, we describe the application of this approach to produce a protein with two distinct chemical functionalites which can be selectively labeled with two fluorescent dyes.

Genetically encoded fluorophenylalanines enable insights into the recognition of lysine trimethylation by an epigenetic reader.

Fluorophenylalanines bearing 2-5 fluorine atoms at the phenyl ring have been genetically encoded by amber codon. Replacement of F59, a phenylalanine residue that is directly involved in interactions with trimethylated K9 of histone H3, in the Mpp8 chromodomain recombinantly with fluorophenylalanines significantly impairs the binding to a K9-trimethylated H3 peptide.

The "π-Clamp" Offers a New Strategy for Site-Selective Protein Modification.

Pentelute and co-workers have identified a small encodable cysteine-containing peptide sequence that allows selective modification with perfluoroaryl probes. This π-clamp requires no external catalyst and is not limited to certain positions within a protein.

Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids.

Using the amber suppression approach, four noncanonical amino acids (ncAAs) were used to replace existing amino acids at four positions in lasso peptide microcin J25 (MccJ25). The lasso peptide biosynthesis enzymes tolerated all four ncAAs and produced antibiotics with efficacy equivalent to wild-type in some cases. Given the rapid expansion of the genetically encoded ncAA pool, this study is the first to demonstrate an expedient method to significantly increase the chemical diversity of lasso peptides.

Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.

The genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (Pyl) at amber codon is achieved by the action of pyrrolysyl-tRNA synthetase (PylRS) together with its cognate tRNA(Pyl). Unlike most aminoacyl-tRNA synthetases, PylRS displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of tRNA(Pyl). These unique but ordinary features of PylRS as an aminoacyl-tRNA synthetase allow the Pyl incorporation machinery to be easily engineered for the genetic incorporation of more than 100 non-canonical amino acids (NCAAs) or α-hydroxy acids into proteins at amber codon and the reassignment of other codons such as ochre UAA, opal UGA, and four-base AGGA codons to code NCAAs.

Genetic incorporation of seven ortho-substituted phenylalanine derivatives.

Seven phenylalanine derivatives with small substitutions were genetically encoded in and mammalian cells at an amber codon using a previously reported, rationally designed pyrrolysyl-tRNA synthetase mutant (PylRS(N346A/C348A)) coupled with tRNA. substitutions of the phenylalanine derivatives reported herein include three halides, methyl, methoxy, nitro, and nitrile. These compounds have the potential for use in multiple biochemical and biophysical applications.