Flexizymes for genetic code reprogramming

Genetic code reprogramming is a method for the reassignment of arbitrary codons from proteinogenic amino acids to nonproteinogenic ones; thus, specific sequences of nonstandard peptides can be ribosomally expressed according to their mRNA templates. Here we describe a protocol that facilitates genetic code reprogramming using flexizymes integrated with a custom-made in vitro translation apparatus, referred to as the flexible in vitro translation (FIT) system. Flexizymes are flexible tRNA acylation ribozymes that enable the preparation of a diverse array of nonproteinogenic acyl-tRNAs. These acyl-tRNAs read vacant codons created in the FIT system, yielding the desired nonstandard peptides with diverse exotic structures, such as N-methyl amino acids, D-amino acids and physiologically stable macrocyclic scaffolds. The facility of the protocol allows a wide variety of applications in the synthesis of new classes of nonstandard peptides with biological functions. Preparation of flexizymes and tRNA used for genetic code reprogramming, optimization of flexizyme reaction conditions and expression of nonstandard peptides using the FIT system can be completed by one person in approximately 1 week. However, once the flexizymes and tRNAs are in hand and reaction conditions are fixed, synthesis of acyl-tRNAs and peptide expression is generally completed in 1 d, and alteration of a peptide sequence can be achieved by simply changing the corresponding mRNA template.     

A highly flexible tRNA acylation method for non-natural polypeptide synthesis

Here we describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the preparation of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows us to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple non-natural amino acids.     

氨酰-tRNA合成酶的研究进展

氨酰-tRNA合成酶催化特异的氨基酸与同源tRNA氨酰化,从而保证了遗传密码翻译的忠实性。这些古老而保守的蛋白质分子除了具有酶的功能外,在哺乳动物细胞中还发现了多种其他功能,具有重要的应用价值。在寻找具有全新作用机制的新抗生素以应对日益严重的抗生素耐药现象过程中,氨酰-tRNA合成酶是细菌蛋白质合成过程中重要的、新颖的靶标,成为关注的重点。定向突变的氨酰-tRNA合成酶可以用来定点掺入非天然氨基酸,扩展蛋白质工程。今后,随着人们对氨酰-tRNA合成酶研究的不断深入,它们还可能用来治疗肿瘤等多种疾病。     

Four-base codon/anticodon strategy and non-enzymatic aminoacylation for protein engineering with non-natural amino acids

Techniques for position-specific incorporation of non-natural amino acids in an in vitro protein synthesizing system are described. First, a PNA-assisted non-enzymatic tRNA aminoacylation with a variety of natural and non-natural amino acids is described. With this technique, one can aminoacylate a specific tRNA simply by adding a preformed amino acid activated ester-PNA conjugate into an in vitro protein biosynthesizing system. Second, the genetic code is expanded by introducing 4-base codons that can be exclusively translated to non-natural amino acids. The most advantageous point of the 4-base codon strategy is to introduce multiple amino acids into specific positions in single proteins by using mutually orthogonal 4-base codons and orthogonal tRNAs. An easy and quick method for preparation of tRNAs possessing 4-base anticodons is also described. Combination of the non-enzymatic aminoacylation and the 4-base codon/anticodon strategy gives an easy and widely applicable technique for incorporating a variety of non-natural amino acids into proteins in vitro.     

Engineering of enzymes using non-natural amino acids

In enzyme engineering, the main targets for enhancing properties are enzyme activity, stereoselective specificity, stability, substrate range, and the development of unique functions. With the advent of genetic code extension technology, non-natural amino acids (nnAAs) are able to be incorporated into proteins in a site-specific or residue-specific manner, which breaks the limit of 20 natural amino acids for protein engineering. Benefitting from this approach, numerous enzymes have been engineered with nnAAs for improved properties or extended functionality. In the present review, we focus on applications and strategies for using nnAAs in enzyme engineering. Notably, approaches to computational modelling of enzymes with nnAAs are also addressed. Finally, we discuss the bottlenecks that currently need to be addressed in order to realise the broader prospects of this genetic code extension technique.     

Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids

The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering.     

Synthesis of biopolymers using genetic code reprogramming

Genetic code reprogramming is a new emerging methodology that enables us to synthesize non-standard peptides containing multiple non-proteinogenic amino acids using translation machinery. This review describes the historical background of this methodology and what distinguishes it from the classical 'nonsense suppression' methodology, followed by a discussion of recent developments in combining this methodology with other compatible technologies. Specifically, we discuss in detail the combination of genetic code reprogramming with flexizymes, de novo tRNA acylation ribozymes that facilitate the charging process of a variety of non-proteinogenic amino acids onto tRNAs bearing designated anticodons, and summarize some of the recent demonstrations of the synthesis of non-standard peptides with cyclic structure or/and altered backbones employing this technology.     

A flexizyme that selectively charges amino acids activated by a water-friendly leaving group

We have developed a new flexizyme (a flexible de novo tRNA acylation ribozyme) system, a pair of amino-derivatized benzyl thioester (ABT) and amino flexizyme (aFx). ABT bearing the ammonium ion was designed to render the acyl-donor substrates better water solubility. Although the previously reported flexizymes (eFx and dFx) did not show acylation activity for the ABT derivatives, a new flexizyme variant aFx, generated by in vitro selection against an amino acid activated ABT, exhibits high selectivity toward those activated ABT. The flexizymes system including aFx, eFx, and dFx enables us to prepare a wide variety of acyl-tRNAs charged with non-proteinogenic amino acids.     

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.     

Development of the genetic code: Insights from a fungal codon reassignment

The high conservation of the genetic code and its fundamental role in genome decoding suggest that its evolution is highly restricted or even frozen. However, various prokaryotic and eukaryotic genetic code alterations, several alternative tRNA-dependent amino acid biosynthesis pathways, regulation of tRNA decoding by diverse nucleoside modifications and recent in vivo incorporation of non-natural amino acids into prokaryotic and eukaryotic proteins, show that the code evolves and is surprisingly flexible. The cellular mechanisms and the proteome buffering capacity that support such evolutionary processes remain unclear. Here we explore the hypothesis that codon misreading and reassignment played fundamental roles in the development of the genetic code and we show how a fungal codon reassignment is enlightening its evolution.     

Is there a twenty third amino acid in the genetic code?

The universal genetic code includes 20 common amino acids. In addition, selenocysteine (Sec) and pyrrolysine (Pyl), known as the twenty first and twenty second amino acids, are encoded by UGA and UAG, respectively, which are the codons that usually function as stop signals. The discovery of Sec and Pyl suggested that the genetic code could be further expanded by reprogramming stop codons. To search for the putative twenty third amino acid, we employed various tRNA identification programs that scanned 16 archaeal and 130 bacterial genomes for tRNAs with anticodons corresponding to the three stop signals. Our data suggest that the occurrence of additional amino acids that are widely distributed and genetically encoded is unlikely.     

Evolution of the Genetic Code by Incorporation of Amino Acids that Improved or Changed Protein Function

Fifty years have passed since the genetic code was deciphered, but how the genetic code came into being has not been satisfactorily addressed. It is now widely accepted that the earliest genetic code did not encode all 20 amino acids found in the universal genetic code as some amino acids have complex biosynthetic pathways and likely were not available from the environment. Therefore, the genetic code evolved as pathways for synthesis of new amino acids became available. One hypothesis proposes that early in the evolution of the genetic code four amino acids—valine, alanine, aspartic acid, and glycine—were coded by GNC codons (N = any base) with the remaining codons being nonsense codons. The other sixteen amino acids were subsequently added to the genetic code by changing nonsense codons into sense codons for these amino acids. Improvement in protein function is presumed to be the driving force behind the evolution of the code, but how improved function was achieved by adding amino acids has not been examined. Based on an analysis of amino acid function in proteins, an evolutionary mechanism for expansion of the genetic code is described in which individual coded amino acids were replaced by new amino acids that used nonsense codons differing by one base change from the sense codons previously used. The improved or altered protein function afforded by the changes in amino acid function provided the selective advantage underlying the expansion of the genetic code. Analysis of amino acid properties and functions explains why amino acids are found in their respective positions in the genetic code.     

Incorporation of non-natural amino acids into proteins

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.     

Protein synthesis: twenty three amino acids and counting

The genetic code can be interpreted during translation as 21 amino acids and three termination signals. Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis.     

Expansion of the genetic code enables design of a novel "gold" class of green fluorescent proteins

Much effort has been dedicated to the design of significantly red shifted variants of the green fluorescent protein (GFP) from Aequoria victora (av). These approaches have been based on classical engineering with the 20 canonical amino acids. We report here an expansion of these efforts by incorporation of an amino substituted variant of tryptophan into the "cyan" GFP mutant, which turned it into a "gold" variant. This variant possesses a red shift in emission unprecedented for any avFP, similar to "red" FPs, but with enhanced stability and a very low aggregation tendency. An increasing number of non-natural amino acids are available for chromophore redesign (by engineering of the genetic code) and enable new general strategies to generate novel classes of tailor-made GFP proteins.     

Structural Basis for the Site-Specific Incorporation of Lysine Derivatives into Proteins

Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the ‘histone code’. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.     

The genetic code--40 years on

The genetic code discovered 40 years ago, consists of 64 triplets (codons) of nucleotides. The genetic code is almost universal. The same codons are assigned to the same amino acids and to the same START and STOP signals in the vast majority of genes in animals, plants, and microorganisms. Each codon encodes for one of the 20 amino acids used in the synthesis of proteins. That produces some redundancy in the code and most of the amino acids being encoded by more than one codon. The two cases have been found where selenocysteine or pyrrolysine, that are not one of the standard 20 is inserted by a tRNA into the growing polypeptide.     

Peptide synthesis with halophenylalanines by thermolysin

Thermolysin was able to catalyze enantioselective peptide synthesis with non-natural amino acids, halophenylalanines. However, the reactivity of thermolysin was considerably influenced by the kind and position of halogen substituents on these analogues. The manner of the recognition of the amino component by the enzyme was different from that of the carboxyl component in the synthesis of peptides with non-natural phenylalanine analogues. The phenomena observed are discussed, based on the kinetic parameters obtained. Correspondence to: A. Tanaka     

Ethynylglycine synthon from Garner’s aldehyde: a useful precursor for the synthesis of non-natural amino acids

Summary. The ethynylglycine synthon, namely (R)-2,2-dimethyl-3-(tert-butoxycarbonyl)-4-ethynyl-oxazolidine, can be obtained through the synthetic elaboration of naturally occurring serine. This compound has been exploited as a helpful and versatile non-racemic building block to be used for the design and synthesis of biologically important compounds, mainly non-natural α-amino acids. Taking advantage of the terminal acetylene moiety several synthetic applications can be designed. Metalation followed by trapping with electrophiles or Cu/Pd catalysed coupling with aromatic halogenides are shown to deliver useful precursors of ethynylglycine derivatives. Additions of bimetallic reagents like stannyl- or silylcuprates are useful entries for the regio- and stereoselective functionalization of the lateral chain, aimed at the synthesis of modified vinylglycine precursors.An overview of our recent work in the field will be given, and the use of ethynylglycine synthon in the synthesis of non-racemic saturated and unsaturated non-natural amino acids will be briefly reviewed.     

Genetic Code Evolution Started with the Incorporation of Glycine,Followed by Other Small Hydrophilic Amino Acids

We propose that glycine was the first amino acid to be incorporated into the genetic code, followed by serine, aspartic and/or glutamic acid—small hydrophilic amino acids that all have codons in the bottom right-hand corner of the standard genetic code table. Because primordial ribosomal synthesis is presumed to have been rudimentary, this stage would have been characterized by the synthesis of short, water-soluble peptides, the first of which would have comprised polyglycine. Evolution of the code is proposed to have occurred by the duplication and mutation of tRNA sequences, which produced a radiation of codon assignment outwards from the bottom right-hand corner. As a result of this expansion, we propose a trend from small hydrophilic to hydrophobic amino acids, with selection for longer polypeptides requiring a hydrophobic core for folding and stability driving the incorporation of hydrophobic amino acids into the code.