Adaptation of an orthogonal archaeal leucyl-tRNA and synthetase pair for four-base,amber, and opal suppression

Recently, it has been shown that an amber suppressor tRNA/aminoacyl-tRNA synthetase pair derived from the tyrosyl-tRNA synthetase of Methanococcus jannaschii can be used to genetically encode unnatural amino acids in response to the amber nonsense codon, TAG. However, we have been unable to modify this pair to decode either the opal nonsense codon, TGA, or the four-base codon, AGGA, limiting us to a 21 amino acid code. To overcome this limitation, we have adapted a leucyl-tRNA synthetase from Methanobacterium thermoautotrophicum and leucyl tRNA derived from Halobacterium sp. NRC-1 as an orthogonal tRNA-synthetase pair in Escherichia coli to decode amber (TAG), opal (TGA), and four-base (AGGA) codons. To improve the efficiency and selectivity of the suppressor tRNA, extensive mutagenesis was performed on the anticodon loop and acceptor stem. The two most significant criteria required for an efficient amber orthogonal suppressor tRNA are a CU(X)XXXAA anticodon loop and the lack of noncanonical or mismatched base pairs in the stem regions. These changes afford only weak suppression of TGA and AGGA. However, this information together with an analysis of sequence similarity of multiple native archaeal tRNA sequences led to efficient, orthogonal suppressors of opal codons and the four-base codon, AGGA. Ultimately, it should be possible to use these additional orthogonal pairs to genetically incorporate multiple unnatural amino acids into proteins.     

Site-specific labeling of proteins with NMR-active unnatural amino acids

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.     

In vitro selection for sense codon suppression

The universal genetic code links the 20 naturally occurring amino acids to the 61 sense codons. Previously, the UAG amber stop codon (a nonsense codon) has been used as a blank in the code to insert natural and unnatural amino acids via nonsense suppression. We have developed a selection methodology to investigate whether the unnatural amino acid biocytin could be incorporated into an mRNA display library at sense codons. In these experiments we probed a single randomized NNN codon with a library of 16 orthogonal, biocytin-acylated tRNAs. In vitro selection for efficient incorporation of the unnatural amino acid resulted in templates containing the GUA codon at the randomized position. This sense suppression occurs via Watson-Crick pairing with similar efficiency to UAG-mediated nonsense suppression. These experiments suggest that sense codon suppression is a viable means to expand the chemical and functional diversity of the genetic code.     

An archaebacteria-derived glutamyl-tRNA synthetase and tRNA pair for unnatural amino acid mutagenesis of proteins in Escherichia coli

The addition of novel amino acids to the genetic code of Escherichia coli involves the generation of an aminoacyl-tRNA synthetase and tRNA pair that is ‘orthogonal’, meaning that it functions independently of the synthetases and tRNAs endogenous to E.coli. The amino acid specificity of the orthogonal synthetase is then modified to charge the corresponding orthogonal tRNA with an unnatural amino acid that is subsequently incorporated into a polypeptide in response to a nonsense or missense codon. Here we report the development of an orthogonal glutamic acid synthetase and tRNA pair. The tRNA is derived from the consensus sequence obtained from a multiple sequence alignment of archaeal tRNA^Glu sequences. The glutamyl-tRNA synthetase is from the achaebacterium Pyrococcus horikoshii. The new orthogonal pair suppresses amber nonsense codons with an efficiency roughly comparable to that of the orthogonal tyrosine pair derived from Methanococcus jannaschii, which has been used to selectively incorporate a variety of unnatural amino acids into proteins in E.coli. Development of the glutamic acid orthogonal pair increases the potential diversity of unnatural amino acid structures that may be incorporated into proteins in E.coli.     

Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells

The genetic code of living organisms has been expanded to allow the site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon UAG. Numerous amino acids have been incorporated including photo-crosslinkers, chemical handles, heavy atoms and post-translational modifications, and this has created new methods for studying biology and developing protein therapeutics and other biotechnological applications. Here we describe a protocol for reprogramming the amino-acid substrate specificity of aminoacyl-tRNA synthetase enzymes that are orthogonal in eukaryotic cells. The resulting aminoacyl-tRNA synthetases aminoacylate an amber suppressor tRNA with a desired unnatural amino acid, but no natural amino acids, in eukaryotic cells. To achieve this change of enzyme specificity, a library of orthogonal aminoacyl-tRNA synthetase is generated and genetic selections are performed on the library in Saccharomyces cerevisiae. The entire protocol, including characterization of the evolved aminoacyl-tRNA synthetase in S. cerevisiae, can be completed in approximately 1 month.     

An efficient protocol for incorporation of an unnatural amino acid in perdeuterated recombinant proteins using glucose-based media

The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR field. Here, we describe a novel protocol for incorporating unnatural amino acids into fully deuterated proteins using glucose-based media (which are relevant to the production, for example, of amino acid-specific methyl-labeled proteins used in the study of large molecular weight systems). The method consists of pre-induction of the pEVOL plasmid encoding the tRNA/aminoacyl-tRNA synthetase pair in a rich, H₂O-based medium prior to exchanging the culture into a D₂O-based medium. Our protocol results in high level of isotopic incorporation (~95%) and retains the high expression level of the target protein observed in Luria–Bertani medium.     

Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.

The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.     

微生物tRNA与密码子系统应用研究进展*

tRNA作为生命中心法则中翻译过程的重要参与分子,其种类、丰度都会对蛋白质的正常合成产生巨大影响。近年来通过对微生物tRNA的结构功能以及合成修饰过程的解析获得诸多启发,开展密码子扩展的研究,实现将非天然氨基酸引入特定位置从而获得新功能蛋白。同时,通过化学合成微生物基因组开展的密码子重编码工作将释放更多的密码子与tRNA用于更加广泛的密码子扩展研究。对微生物tRNA与密码子系统在合成生物学中的最新应用研究进展进行了综述,并讨论其未来的发展趋势。     

Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: minimizing misacylation

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure-function studies, incorporation of biophysical probes, and determining protein-protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells.     

Complete set of orthogonal 21st aminoacyl-tRNA synthetase-amber, ochre and opal suppressor tRNA pairs: concomitant suppression of three different termination codons in an mRNA in mammalian cells

We describe the generation of a complete set of orthogonal 21st synthetase-amber, ochre and opal suppressor tRNA pairs including the first report of a 21st synthetase-ochre suppressor tRNA pair. We show that amber, ochre and opal suppressor tRNAs, derived from Escherichia coli glutamine tRNA, suppress UAG, UAA and UGA termination codons, respectively, in a reporter mRNA in mammalian cells. Activity of each suppressor tRNA is dependent upon the expression of E.coli glutaminyl-tRNA synthetase, indicating that none of the suppressor tRNAs are aminoacylated by any of the twenty aminoacyl-tRNA synthetases in the mammalian cytoplasm. Amber, ochre and opal suppressor tRNAs with a wide range of activities in suppression (increases of up to 36, 156 and 200-fold, respectively) have been generated by introducing further mutations into the suppressor tRNA genes. The most active suppressor tRNAs have been used in combination to concomitantly suppress two or three termination codons in an mRNA. We discuss the potential use of these 21st synthetase-suppressor tRNA pairs for the site-specific incorporation of two or, possibly, even three different unnatural amino acids into proteins and for the regulated suppression of amber, ochre and opal termination codons in mammalian cells.     

Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs

The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an extrachromosomal array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature dependencies similar to those of endogenous amber suppression. Uaa incorporation efficiency was improved by impairing the nonsense-mediated mRNA decay pathway through knockdown of smg-1. We have generated stable transgenic worms capable of genetically encoding Uaas, enabling Uaa exploitation to address complex biological problems within a metazoan. We anticipate our strategies will be generally extendable to other multicellular organisms.     

Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA^Tyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue–white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA^Tyr.     

Genetic incorporation of unnatural amino acids into proteins in mammalian cells

We developed a general approach that allows unnatural amino acids with diverse physicochemical and biological properties to be genetically encoded in mammalian cells. A mutant Escherichia coli aminoacyl-tRNA synthetase (aaRS) is first evolved in yeast to selectively aminoacylate its tRNA with the unnatural amino acid of interest. This mutant aaRS together with an amber suppressor tRNA from Bacillus stearothermophilus is then used to site-specifically incorporate the unnatural amino acid into a protein in mammalian cells in response to an amber nonsense codon. We independently incorporated six unnatural amino acids into GFP expressed in CHO cells with efficiencies up to 1 mug protein per 2 x 10(7) cells; mass spectrometry confirmed a high translational fidelity for the unnatural amino acid. This methodology should facilitate the introduction of biological probes into proteins for cellular studies and may ultimately facilitate the synthesis of therapeutic proteins containing unnatural amino acids in mammalian cells.     

Site-specific insertion of spin-labeled L-amino acids in Xenopus oocytes

Site-specific insertion of modified amino acids in proteins expressed in living cells is an emerging field holding great promise for elucidating protein structure-function relationships, expression levels, localization, and activation states in a complex milieu. To evaluate the efficiency of amino acids modified to carry either a nitroxide spin probe or a fluorescence probe, we have developed a screen using the levels of functional luciferase protein expressed in Xenopus oocytes. Natural and modified amino acids were targeted to position 14 in firefly luciferase using an amber mutation or introducing the four-codon nucleotide GGGU. Using the amber stop codon, the incorporation efficiencies of injected tRNA charged with the native phenylalanine residue, a fluorescent NBD-alanine, or nitroxide-labeled cysteine and tyrosine amino acids ranged from 1% to 18%. While the NBD-amino acid derivative gave higher incorporation levels, the EPR signals from the spin-labeled amino acids allow for the direct assessment of aminoacylation extent and stability. Applying the four-base codon for the first time in Xenopus oocytes, we found the incorporation efficiencies were significantly lowered compared to results using the three-base amber codon. The studies presented here provide quantitative assessment of protein expression levels when using nonsense suppression to site-specifically label proteins with spectroscopic probes in oocytes. Finally, the effect of a 77-base RNA aptamer known to inhibit the eucaryotic release factor of protein synthesis was tested for its influence on nonsense incorporation in Xenopus oocytes. The combination of A34 and charged suppressor tRNA produced a 3-fold increase in the expressed TAG(14)-luciferase level, compared to the use of charged suppressor tRNA alone.     

Introduction of specialty functions by the position-specific incorporation of nonnatural amino acids into proteins through four-base codon/anticodon pairs

Position-specific incorporation of nonnatural amino acids into proteins (nonnatural mutagenesis) via an in vitro protein synthesizing system was applied to incorporate a variety of amino acids carrying specialty side groups. A list of nonnatural amino acids thus far successfully incorporated through in vitro translation systems is presented. The position of nonnatural amino acid incorporation was directed by four-base codon/anticodon pairs such as CGGG/CCCG and AGGU/ACCU. The four-base codon strategy was more efficient than the amber codon strategy and could incorporate multiple nonnatural amino acids into single proteins. This multiple mutagenesis will find wide applications, especially in building paths of electron transfer on proteins. The extension of translation systems by the introduction of nonnatural amino acids, four-base codon/anticodon pairs, orthogonal tRNAs, and artificial aminoacyl tRNA synthetases, is a promising approach towards the creation of "synthetic microorganisms" with specialty functions.     

Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons

A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (Ψ) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. Ψ differs from uridine by a single N1H group at the C5 position; how Ψ suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, we provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Our analysis applies crystal structures of ribosomes in varying states of translation to consider weakened interaction of Ψ with release factor; thermodynamic and geometric considerations of the codon-anticodon base pairs to rank and to eliminate mRNA-tRNA pairs; the mechanism of fidelity check of the codon-anticodon pairing by the ribosome to evaluate noncanonical codon-anticodon base pairs and the role of water. We also consider certain tRNA modifications that interfere with the Ψ-coordinated water in the major groove of the codon-anticodon mini-helix. Our analysis of nonsense codons enables prediction of potential decoding properties for Ψ-modified sense codons, such as decoding ΨUU potentially as Cys and Tyr. Our results provide molecular rationale for the remarkable dynamics of ribosome decoding and insights on possible reprogramming of the genetic code using mRNA modifications.     

Rewiring translation - Genetic code expansion and its applications

With few minor variations, the genetic code is universal to all forms of life on our planet. It is difficult to imagine that one day organisms might exist that use an entirely different code to translate the information of their genome. Recent developments in the field of synthetic biology, however, have opened the gate to their creation. The genetic code of several organisms has been expanded by the heterologous expression of evolved aminoacyl-tRNA synthetase/tRNA(CUA) pairs that mediate the incorporation of unnatural amino acids in response to amber codons. These UAAs introduce exciting new features into proteins, such as spectroscopic probes, UV-inducible crosslinkers, and functional groups for bioorthogonal conjugations or posttranslational modifications. Orthogonal ribosomes provide a parallel translational machinery in Escherichia coli that has lost its evolutionary constraints. Evolved variants of these ribosomes translate amber or quadruplet codons with massively enhanced efficiency. Here, I review these recent developments emphasizing their tremendous potential to facilitate biochemical and cell biological studies.     

Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment

Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha factor of Saccharomyces cerevisiae. This receptor-pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl- l-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid residues in Ste2p with Bpa by engineering amber TAG stop codons into STE2 encoded on a plasmid. Several of the expressed Bpa-substituted Ste2p receptors exhibited high-affinity ligand binding, and incorporation of Bpa into Ste2p influenced biological activity as measured by growth arrest of whole cells in response to alpha factor. We found that, at concentrations of 0.1-0.5 mM, a dipeptide containing Bpa could be used to enhance delivery of Bpa into the cell, while at 2 mM, both dipeptide and Bpa were equally effective. The application of a peptide delivery system for unnatural amino acids will extend the use of the unnatural amino acid replacement methodology to amino acids that are impermeable to yeast. Incorporation of Bpa into Ste2p was verified by mass spectrometric analysis, and two Bpa-Ste2p mutants were able to selectively capture alpha factor into the ligand-binding site after photoactivation. To our knowledge, this is the first experimental evidence documenting an unnatural amino acid replacement in a GPCR expressed in its native environment and the use of a mutated receptor to photocapture a peptide ligand.