A general approach for the generation of orthogonal tRNAs

BACKGROUND: The addition of new amino acids to the genetic code of Escherichia coli requires an orthogonal suppressor tRNA that is uniquely acylated with a desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. A tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair imported from Methanococcus jannaschii can be used to generate such a pair. In vivo selections have been developed for selecting mutant suppressor tRNAs with enhanced orthogonality, which can be used to site-specifically incorporate unnatural amino acids into proteins in E. coli. RESULTS: A library of amber suppressor tRNAs derived from M. jannaschii tRNA(Tyr) was generated. tRNA(Tyr)(CUA)s that are substrates for endogenous E. coli aminoacyl-tRNA synthetases were deleted from the pool by a negative selection based on suppression of amber nonsense mutations in the barnase gene. The remaining tRNA(Tyr)(CUA)s were then selected for their ability to suppress amber nonsense codons in the beta-lactamase gene in the presence of the cognate M. jannaschii tyrosyl-tRNA synthetase (TyrRS). Four mutant suppressor tRNAs were selected that are poorer substrates for E. coli synthetases than M. jannaschii tRNA(Tyr)(CUA), but still can be charged efficiently by M. jannaschii TyrRS. CONCLUSIONS: The mutant suppressor tRNA(Tyr)(CUA) together with the M. jannaschii TyrRS is an excellent orthogonal tRNA-synthetase pair for the in vivo incorporation of unnatural amino acids into proteins. This general approach may be expanded to generate additional orthogonal tRNA-synthetase pairs as well as probe the interactions between tRNAs and their cognate synthetases.     

Progress toward an expanded eukaryotic genetic code

Expanding the eukaryotic genetic code to include unnatural amino acids with novel properties would provide powerful tools for manipulating protein function in eukaryotic cells. Toward this goal, a general approach with potential for isolating aminoacyl-tRNA synthetases that incorporate unnatural amino acids with high fidelity into proteins in Saccharomyces cerevisiae is described. The method is based on activation of GAL4-responsive HIS3, URA3, or lacZ reporter genes by suppression of amber codons in GAL4. The optimization of GAL4 reporters is described, and the positive and negative selection of active Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA(CUA) is demonstrated. Importantly, both selections can be performed on a single cell and with a range of stringencies. This method will facilitate the isolation of a range of aminoacyl-tRNA synthetase (aaRS)/tRNA(CUA) activities from large libraries of mutant synthetases.     

A New Orthogonal Suppressor tRNA/Aminoacyl‐tRNA Synthetase Pair for Evolving an Organism with an Expanded Genetic Code

Several steps have been completed toward the development of a method for the site‐specific incorporation of unnatural amino acids into proteins in vivo. Our approach consists of the generation of amber suppressor tRNA/aminoacyl‐tRNA synthetase pairs that are orthogonal to all Escherichia coli endogenous tRNA/synthetase pairs, followed by directed evolution of the orthogonal aminoacyl‐tRNA synthetases to alter their amino‐acid specificities. A new orthogonal suppressor tRNA/aminoacyl‐tRNA synthetase pair in E. coli has been derived from the Saccharomyces cerevisiae tRNA^Asp and aspartyl‐tRNA synthetase, and the in vitro and in vivo characteristics of this pair were determined. Two different antibiotic resistance selections were compared using this novel pair in an effort to develop a tunable positive selection for a mutant synthetase capable of charging its cognate suppressor tRNA with an unnatural amino acid.     

Adding L-3-(2-Naphthyl)alanine to the genetic code of E. coli

An unnatural amino acid, L-3-(2-naphthyl)alanine, has been site-specifically incorporated into proteins in Escherichia coli. An orthogonal aminoacyl-tRNA synthetase was evolved that uniquely aminoacylates the unnatural amino acid onto an orthogonal amber suppressor tRNA, which delivers the acylated amino acid in response to an amber nonsense codon with translational fidelity greater than 99%. This result, together with the in vivo site-specific incorporation of O-methyl-L-tyrosine reported previously, demonstrate that this methodology may be applicable to a host of amino acids. The expansion of the genetic code to include amino acids beyond the common 20 would provide an opportunity to better understand and possibly enhance protein (and perhaps organismal) function.     

Rational design to block amino acid editing of a tRNA synthetase

Investigations in chemical biology have focused on the synthesis of custom-designed proteins with site-specific incorporation of novel amino acids. Their success requires stable production of misacylated tRNAs. Utilization of aminoacyl-tRNA synthetases has been hindered because of enzyme molecular recognition mechanisms that ensure high fidelity of protein synthesis. Leucyl-tRNA synthetase naturally misaminoacylates chemically diverse standard and nonstandard amino acids, but contains a separate amino acid editing active site to hydrolyze incorrectly mischarged tRNAs. In this work, a rational mutagenesis design to block enzyme editing is described and involves substitution of bulky amino acids into the amino acid binding pocket of the hydrolytic active site. These engineered enzymes stably misacylated isoleucine to tRNALeu. The use of these mutant leucyl-tRNA synthetases has the potential to produce pools of mischarged tRNAs that are linked to nonstandard amino acids for in vitro translation. In addition, since many of the leucyl-tRNA synthetases do not interact with or rely upon the tRNA anticodon for identity, these enzymes may offer an excellent scaffold for the development of orthogonal tRNA synthetase/tRNA pairs that can potentially be used to customize protein synthesis.     

Proximity‐Enabled Protein Crosslinking through Genetically Encoding Haloalkane Unnatural Amino Acids

The selective generation of covalent bonds between and within proteins would provide new avenues for studying protein function and engineering proteins with new properties. New covalent bonds were genetically introduced into proteins by enabling an unnatural amino acid (Uaa) to selectively react with a proximal natural residue. This proximity‐enabled bioreactivity was expanded to a series of haloalkane Uaas. Orthogonal tRNA/synthetase pairs were evolved to incorporate these Uaas, which only form a covalent thioether bond with cysteine when positioned in close proximity. By using the Uaa and cysteine, spontaneous covalent bond formation was demonstrated between an affibody and its substrate Z protein, thereby leading to irreversible binding, and within the affibody to increase its thermostability. This strategy of proximity‐enabled protein crosslinking (PEPC) may be generally expanded to target different natural amino acids, thus providing diversity and flexibility in covalent bond formation for protein research and protein engineering.     

Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair

We report the discovery of a simple system through which variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs created in Escherichia coli can be used to expand the genetic code of Saccharomyces cerevisiae. In the process we have solved the key challenges of producing a functional tRNA(CUA Pyl) in yeast and discovered a pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pair that is orthogonal in yeast. Using our approach we have incorporated an alkyne-containing amino acid for click chemistry, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid and a photo-cross-linking amino acid into proteins in yeast. Extensions of our approach will allow the growing list of useful amino acids that have been incorporated in E. coli with variant pyrrolysyl-tRNA synthetase/tRNA(CUA Pyl) pairs to be site-specifically incorporated into proteins in yeast.     

Structural characterization of a p-acetylphenylalanyl aminoacyl-tRNA synthetase

It has been recently shown that orthogonal tRNA/aminoacyl-tRNA synthetase pairs can be evolved to allow genetic incorporation of unnatural amino acids into proteins in both prokaryotes and eukaryotes. Here we describe the crystal structure of an evolved aminoacyl-tRNA synthetase that charges the unnatural amino acid p-acetylphenylalanine. Molecular recognition is due to altered hydrogen bonding and packing interactions with bound substrate that result from changes in both side-chain and backbone conformation.     

Reprogramming the genetic code: from triplet to quadruplet codes

The genetic code of cells is near-universally triplet, and since many ribosomal mutations are lethal, changing the cellular ribosome to read nontriplet codes is challenging. Herein we review work on the incorporation of unnatural amino acids into proteins in response to quadruplet codons, and the creation of an orthogonal translation system in the cell that uses an evolved orthogonal ribosome to efficiently direct the incorporation of unnatural amino acids in response to quadruplet codons. Using this system multiple distinct unnatural amino acids have been incorporated and used to genetically program emergent properties into recombinant proteins. Extension of approaches to incorporate multiple unnatural amino acids may allow the combinatorial biosynthesis of materials and therapeutics, and drive investigations into whether life with additional genetically encoded polymers can evolve to perform functions that natural biological systems cannot.     

In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy

In vivo incorporation of isotopically labeled unnatural amino acids into large proteins drastically reduces the complexity of nuclear magnetic resonance (NMR) spectra. Incorporation is accomplished by coexpressing an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid added to the media and the protein of interest with a TAG amber codon at the desired incorporation site. To demonstrate the utility of this approach for NMR studies, 2-amino-3-(4-(trifluoromethoxy)phenyl)propanoic acid (OCF 3Phe), (13)C/(15)N-labeled p-methoxyphenylalanine (OMePhe), and (15)N-labeled o-nitrobenzyl-tyrosine (oNBTyr) were incorporated individually into 11 positions around the active site of the 33 kDa thioesterase domain of human fatty acid synthase (FAS-TE). In the process, a novel tRNA synthetase was evolved for OCF 3Phe. Incorporation efficiencies and FAS-TE yields were improved by including an inducible copy of the respective aminoacyl-tRNA synthetase gene on each incorporation plasmid. Using only between 8 and 25 mg of unnatural amino acid, typically 2 mg of FAS-TE, sufficient for one 0.1 mM NMR sample, were produced from 50 mL of Escherichia coli culture grown in rich media. Singly labeled protein samples were then used to study the binding of a tool compound. Chemical shift changes in (1)H-(15)N HSQC, (1)H-(13)C HSQC, and (19)F NMR spectra of the different single site mutants consistently identified the binding site and the effect of ligand binding on conformational exchange of some of the residues. OMePhe or OCF 3Phe mutants of an active site tyrosine inhibited binding; incorporating (15)N-Tyr at this site through UV-cleavage of the nitrobenzyl-photocage from oNBTyr re-established binding. These data suggest not only robust methods for using unnatural amino acids to study large proteins by NMR but also establish a new avenue for the site-specific labeling of proteins at individual residues without altering the protein sequence, a feat that can currently not be accomplished with any other method.     

Generation of a bacterium with a 21 amino acid genetic code

We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.     

A Facile System for Encoding Unnatural Amino Acids in Mammalian Cells

A shuttle system has been developed to genetically encode unnatural amino acids in mammalian cells using aminoacyl‐tRNA synthetases (aaRSs) evolved in E. coli. A pyrrolysyl‐tRNA synthetase (PylRS) mutant was evolved in E. coli that selectively aminoacylates a cognate nonsense suppressor tRNA with a photocaged lysine derivative. Transfer of this orthogonal tRNA–aaRS pair into mammalian cells made possible the selective incorporation of this unnatural amino acid into proteins.

     

Ribosomal synthesis of unnatural peptides

Combinatorial libraries of non-biological polymers and drug-like peptides could in principle be synthesized from unnatural amino acids by exploiting the broad substrate specificity of the ribosome. The ribosomal synthesis of such libraries would allow rare functional molecules to be identified using technologies developed for the in vitro selection of peptides and proteins. Here, we use a reconstituted E. coli translation system to simultaneously re-assign 35 of the 61 sense codons to 12 unnatural amino acid analogues. This reprogrammed genetic code was used to direct the synthesis of a single peptide containing 10 different unnatural amino acids. This system is compatible with mRNA-display, enabling the synthesis of unnatural peptide libraries of 10(14) unique members for the in vitro selection of functional unnatural molecules. We also show that the chemical space sampled by these libraries can be expanded using mutant aminoacyl-tRNA synthetases for the incorporation of additional unnatural amino acids or by the specific posttranslational chemical derivitization of reactive groups with small molecules. This system represents a first step toward a platform for the synthesis by enzymatic tRNA aminoacylation and ribosomal translation of cyclic peptides comprised of unnatural amino acids that are similar to the nonribosomal peptides.     

A biosynthetic route to photoclick chemistry on proteins

Light-induced chemical reactions exist in nature, regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical "handle" for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.     

Improving nature's enzyme active site with genetically encoded unnatural amino acids

The ability to site-specifically incorporate a diverse set of unnatural amino acids (>30) into proteins and quickly add new structures of interest has recently changed our approach to protein use and study. One important question yet unaddressed with unnatural amino acids (UAAs) is whether they can improve the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the >30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active site and a >2.3-fold improvement over the best possible natural amino acid. Because immense structural and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to improve enzyme active sites and tune a site to multiple substrates.     

The incorporation of a photoisomerizable amino acid into proteins in E. coli

An orthogonal aminoacyl tRNA synthetase/tRNA pair has been evolved that allows the incorporation of the photoisomerizable amino acid phenylalanine-4'-azobenzene (AzoPhe) into proteins in E. coli in response to the amber nonsense codon. Further, we show that AzoPhe can be used to photoregulate the binding affinity of catabolite activator protein to its promoter. The ability to selectively incorporate AzoPhe into proteins at defined sites should make it possible to regulate a variety of biological processes with light, including enzyme, receptor, and ion channel activity.