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Special Issue "Expanding and Reprogramming the Genetic Code"

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Biology".

Deadline for manuscript submissions: closed (31 March 2019)

Special Issue Editor

Guest Editor
Dr. Kensaku Sakamoto

Laboratory for Nonnatural Amino Acid Technology, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan
Website | E-Mail
Interests: genetic code expansion; codon reassignment; tRNA; aminoacyl-tRNA synthetase

Special Issue Information

Dear Colleagues,

The genetic code defines the fundamental rule of translating genetic information into proteins, and is presumably unchanged since its establishment billions of years ago. The 64 base triplets (codons) specify 22 amino acids and translation stops. The additional encoding of new amino acids requires the acquisition of specific molecular machinery and the adjustment of the codon usage in the organism to avoid lethal effects. Due to advanced knowledge and biotechnology, these hurdles have partly been overcome, making substantial progress toward the extensive reprogramming of the genetic code in living cells. The genetic code has successfully been modified, even in animals. Engineered codes could produce proteins, molecular systems/pathways, and organisms never realized in the history of life, through either artificial design or the autonomous evolution of host cells. The currently-available amino acids are almost exclusively confined to tyrosine and pyrrolysine derivatives, and further expansion of the amino-acid repertoire relies on the engineering of new tRNA–aminoacyl-tRNA synthetase pairs. The developed pairs can be used for redefining the meaning of multiple codons simultaneously, after a genome-wide rearrangement in codon usage makes this change viable. Only a few codons are currently useful. On the other hand, expanded codes have found various applications in basic science and industry, and enabled the exploration of biosystems supported by non-natural proteins. This Special Issue will cover original reports and review articles on method developments, applications, and future perspectives of genetic code expansion, as well as natural variations in translational molecules and machinery, which can inspire new directions of engineering.

Dr. Kensaku Sakamoto
Guest Editor

Manuscript Submission Information

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Keywords

  • unnatural amino acids
  • pyrrolysine
  • tRNA
  • aminoacyl-tRNA synthetase
  • quadruplet codons
  • orthogonal ribosome
  • codon reassignment
  • codon usage
  • animals
  • bacterial fitness
  • cell-free translation
  • photo-crosslink
  • chemical conjugate
  • protein engineering

Published Papers (16 papers)

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Research

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Open AccessCommunication
Expanding the Zebrafish Genetic Code through Site-Specific Introduction of Azido-lysine, Bicyclononyne-lysine, and Diazirine-lysine
Int. J. Mol. Sci. 2019, 20(10), 2577; https://doi.org/10.3390/ijms20102577
Received: 9 May 2019 / Revised: 22 May 2019 / Accepted: 23 May 2019 / Published: 26 May 2019
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Abstract
Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs [...] Read more.
Site-specific incorporation of un-natural amino acids (UNAA) is a powerful approach to engineer and understand protein function. Site-specific incorporation of UNAAs is achieved through repurposing the amber codon (UAG) as a sense codon for the UNAA, using a tRNACUA that base pairs with an UAG codon in the mRNA and an orthogonal amino-acyl tRNA synthetase (aaRS) that charges the tRNACUA with the UNAA. Here, we report an expansion of the zebrafish genetic code to incorporate the UNAAs, azido-lysine (AzK), bicyclononyne-lysine (BCNK), and diazirine-lysine (AbK) into green fluorescent protein (GFP) and glutathione-s-transferase (GST). We also present proteomic evidence for UNAA incorporation into GFP. Our work sets the stage for the use of AzK, BCNK, and AbK introduction into proteins as a means to investigate and engineer their function in zebrafish. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessArticle
Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine
Int. J. Mol. Sci. 2019, 20(9), 2343; https://doi.org/10.3390/ijms20092343
Received: 16 April 2019 / Revised: 7 May 2019 / Accepted: 9 May 2019 / Published: 11 May 2019
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Abstract
Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5–7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. [...] Read more.
Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5–7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessArticle
In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation
Int. J. Mol. Sci. 2019, 20(9), 2299; https://doi.org/10.3390/ijms20092299
Received: 18 April 2019 / Revised: 5 May 2019 / Accepted: 7 May 2019 / Published: 9 May 2019
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Abstract
In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis [...] Read more.
In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins—a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessArticle
Characterization and Scaled-Up Production of Azido-Functionalized Silk Fiber Produced by Transgenic Silkworms with an Expanded Genetic Code
Int. J. Mol. Sci. 2019, 20(3), 616; https://doi.org/10.3390/ijms20030616
Received: 21 December 2018 / Revised: 24 January 2019 / Accepted: 28 January 2019 / Published: 31 January 2019
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Abstract
The creation of functional materials from renewable resources has attracted much interest. We previously reported on the genetic code expansion of the domesticated silkworm Bombyx mori to functionalize silk fiber with synthetic amino acids such as 4-azido-L-phenylalanine (AzPhe). The azido groups act as [...] Read more.
The creation of functional materials from renewable resources has attracted much interest. We previously reported on the genetic code expansion of the domesticated silkworm Bombyx mori to functionalize silk fiber with synthetic amino acids such as 4-azido-L-phenylalanine (AzPhe). The azido groups act as selective handles for biorthogonal chemical reactions. Here we report the characterization and scaled-up production of azido-functionalized silk fiber for textile, healthcare, and medical applications. To increase the productivity of azido-functionalized silk fiber, the original transgenic line was hybridized with a high silk-producing strain. The F1 hybrid produced circa 1.5 times more silk fibroin than the original transgenic line. The incorporation efficiency of AzPhe into silk fibroin was retained after hybridization. The tensile properties of the azido-functionalized silk fiber were equal to those of normal silk fiber. Scaled-up production of the azido-functionalized silk fiber was demonstrated by rearing circa 1000 transgenic silkworms. Differently-colored fluorescent silk fibers were successfully prepared by click chemistry reactions, demonstrating the utility of the azido-functionalized silk fiber for developing silk-based materials with desired functions. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessArticle
Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins
Int. J. Mol. Sci. 2019, 20(3), 492; https://doi.org/10.3390/ijms20030492
Received: 19 December 2018 / Revised: 16 January 2019 / Accepted: 21 January 2019 / Published: 24 January 2019
Cited by 1 | PDF Full-text (2482 KB) | HTML Full-text | XML Full-text
Abstract
Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is [...] Read more.
Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessArticle
Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures
Int. J. Mol. Sci. 2019, 20(1), 92; https://doi.org/10.3390/ijms20010092
Received: 29 November 2018 / Revised: 19 December 2018 / Accepted: 22 December 2018 / Published: 26 December 2018
Cited by 1 | PDF Full-text (993 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the [...] Read more.
The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNAPyl. First, we constructed a naïve hybrid between archaeal tRNAPyl and tRNATyr, which consisted of the acceptor and T stems of tRNATyr and the other parts of tRNAPyl. This hybrid tRNA efficiently translated the UAG codon to 3-iodotyrosine in Escherichia coli cells, when paired with a variant of the archaeal tyrosyl-tRNA synthetase. The amber suppression efficiency was slightly lower than that of the “bench-mark” archaeal tRNATyr suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNATyr: ZtRNA had an augmented D (auD) helix in a noncanonical form and the D and T loops bound by the standard tertiary base pairs, and YtRNA had a canonical auD helix and non-standard interloop interactions. It was then suggested that the ZtRNA scaffold could also support the glycylation and glutaminylation of tRNA. The synthetic diversity of tRNA would help create new tRNA–aminoacyl-tRNA synthetase pairs for reprogramming the genetic code. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessCommunication
Rapid Identification of Functional Pyrrolysyl-tRNA Synthetases via Fluorescence-Activated Cell Sorting
Int. J. Mol. Sci. 2019, 20(1), 29; https://doi.org/10.3390/ijms20010029
Received: 23 November 2018 / Revised: 13 December 2018 / Accepted: 20 December 2018 / Published: 21 December 2018
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Abstract
The orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants [...] Read more.
The orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and their variants have provided powerful tools for expanding the genetic code to allow for engineering of proteins with augmented structure and function not present in Nature. To expedite the discovery of novel pyrrolysyl-tRNA synthetase (PylRS) variants that can charge non-natural amino acids into proteins site-specifically, herein we report a streamlined protocol for rapid construction of the pyrrolysyl-tRNA synthetase library, selection of the functional PylRS mutants using fluorescence-activated cell sorting, and subsequent validation of the selected PylRS mutants through direct expression of the fluorescent protein reporter using a single bacterial strain. We expect that this protocol should be generally applicable to rapid identification of the functional PylRS mutants for charging a wide range of non-natural amino acids into proteins. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessCommunication
Presyncodon, a Web Server for Gene Design with the Evolutionary Information of the Expression Hosts
Int. J. Mol. Sci. 2018, 19(12), 3872; https://doi.org/10.3390/ijms19123872
Received: 15 November 2018 / Revised: 29 November 2018 / Accepted: 3 December 2018 / Published: 4 December 2018
Cited by 1 | PDF Full-text (929 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
In the natural host, most of the synonymous codons of a gene have been evolutionarily selected and related to protein expression and function. However, for the design of a new gene, most of the existing codon optimization tools select the high-frequency-usage codons and [...] Read more.
In the natural host, most of the synonymous codons of a gene have been evolutionarily selected and related to protein expression and function. However, for the design of a new gene, most of the existing codon optimization tools select the high-frequency-usage codons and neglect the contribution of the low-frequency-usage codons (rare codons) to the expression of the target gene in the host. In this study, we developed the method Presyncodon, available in a web version, to predict the gene code from a protein sequence, using built-in evolutionary information on a specific expression host. The synonymous codon-usage pattern of a peptide was studied from three genomic datasets (Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae). Machine-learning models were constructed to predict a selection of synonymous codons (low- or high-frequency-usage codon) in a gene. This method could be easily and efficiently used to design new genes from protein sequences for optimal expression in three expression hosts (E. coli, B. subtilis, and S. cerevisiae). Presyncodon is free to academic and noncommercial users; accessible at http://www.mobioinfor.cn/presyncodon_www/index.html. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessArticle
Differences in Codon Usage Bias between Photosynthesis-Related Genes and Genetic System-Related Genes of Chloroplast Genomes in Cultivated and Wild Solanum Species
Int. J. Mol. Sci. 2018, 19(10), 3142; https://doi.org/10.3390/ijms19103142
Received: 29 August 2018 / Revised: 30 September 2018 / Accepted: 4 October 2018 / Published: 12 October 2018
Cited by 1 | PDF Full-text (2514 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Solanum is one of the largest genera, including two important crops—potato (Solanum tuberosum) and tomato (Solanum lycopersicum). In this study we compared the chloroplast codon usage bias (CUB) among 12 Solanum species, between photosynthesis-related genes (Photo-genes) and genetic system-related [...] Read more.
Solanum is one of the largest genera, including two important crops—potato (Solanum tuberosum) and tomato (Solanum lycopersicum). In this study we compared the chloroplast codon usage bias (CUB) among 12 Solanum species, between photosynthesis-related genes (Photo-genes) and genetic system-related genes (Genet-genes), and between cultivated species and wild relatives. The Photo-genes encode proteins for photosystems, the photosynthetic electron transport chain, and RuBisCO, while the Genet-genes encode proteins for ribosomal subunits, RNA polymerases, and maturases. The following findings about the Solanum chloroplast genome CUB were obtained: (1) the nucleotide composition, gene expression, and selective pressure are identified as the main factors affecting chloroplast CUB; (2) all these 12 chloroplast genomes prefer A/U over G/C and pyrimidines over purines at the third-base of codons; (3) Photo-genes have higher codon adaptation indexes than Genet-genes, indicative of a higher gene expression level and a stronger adaptation of Photo-genes; (4) gene function is the primary factor affecting CUB of Photo-genes but not Genet-genes; (5) Photo-genes prefer pyrimidine over purine, whereas Genet-genes favor purine over pyrimidine, at the third position of codons; (6) Photo-genes are mainly affected by the selective pressure, whereas Genet-genes are under the underlying mutational bias; (7) S. tuberosum is more similar with Solanum commersonii than with Solanum bulbocastanum; (8) S. lycopersicum is greatly different from the analyzed seven wild relatives; (9) the CUB in codons for valine, aspartic acid, and threonine are the same between the two crop species, S. tuberosum and S. lycopersicum. These findings suggest that the chloroplast CUB contributed to the differential requirement of gene expression activity and function between Photo-genes and Genet-genes and to the performance of cultivated potato and tomato. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessArticle
Comprehensive Analysis of Codon Usage on Rabies Virus and Other Lyssaviruses
Int. J. Mol. Sci. 2018, 19(8), 2397; https://doi.org/10.3390/ijms19082397
Received: 2 July 2018 / Revised: 8 August 2018 / Accepted: 10 August 2018 / Published: 14 August 2018
Cited by 4 | PDF Full-text (1952 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Rabies virus (RABV) and other lyssaviruses can cause rabies and rabies-like diseases, which are a persistent public health threat to humans and other mammals. Lyssaviruses exhibit distinct characteristics in terms of geographical distribution and host specificity, indicative of a long-standing diversification to adapt [...] Read more.
Rabies virus (RABV) and other lyssaviruses can cause rabies and rabies-like diseases, which are a persistent public health threat to humans and other mammals. Lyssaviruses exhibit distinct characteristics in terms of geographical distribution and host specificity, indicative of a long-standing diversification to adapt to the environment. However, the evolutionary diversity of lyssaviruses, in terms of codon usage, is still unclear. We found that RABV has the lowest codon usage bias among lyssaviruses strains, evidenced by its high mean effective number of codons (ENC) (53.84 ± 0.35). Moreover, natural selection is the driving force in shaping the codon usage pattern of these strains. In summary, our study sheds light on the codon usage patterns of lyssaviruses, which can aid in the development of control strategies and experimental research. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Review

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Open AccessReview
Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases
Int. J. Mol. Sci. 2019, 20(9), 2294; https://doi.org/10.3390/ijms20092294
Received: 15 April 2019 / Revised: 29 April 2019 / Accepted: 7 May 2019 / Published: 9 May 2019
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Abstract
Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) [...] Read more.
Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessReview
Aminoacyl-tRNA Synthetases and tRNAs for an Expanded Genetic Code: What Makes them Orthogonal?
Int. J. Mol. Sci. 2019, 20(8), 1929; https://doi.org/10.3390/ijms20081929
Received: 1 April 2019 / Revised: 16 April 2019 / Accepted: 17 April 2019 / Published: 19 April 2019
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Abstract
In the past two decades, tRNA molecules and their corresponding aminoacyl-tRNA synthetases (aaRS) have been extensively used in synthetic biology to genetically encode post-translationally modified and unnatural amino acids. In this review, we briefly examine one fundamental requirement for the successful application of [...] Read more.
In the past two decades, tRNA molecules and their corresponding aminoacyl-tRNA synthetases (aaRS) have been extensively used in synthetic biology to genetically encode post-translationally modified and unnatural amino acids. In this review, we briefly examine one fundamental requirement for the successful application of tRNA/aaRS pairs for expanding the genetic code. This requirement is known as “orthogonality”—the ability of a tRNA and its corresponding aaRS to interact exclusively with each other and avoid cross-reactions with additional types of tRNAs and aaRSs in a given organism. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessReview
Translational Control using an Expanded Genetic Code
Int. J. Mol. Sci. 2019, 20(4), 887; https://doi.org/10.3390/ijms20040887
Received: 19 December 2018 / Revised: 14 February 2019 / Accepted: 15 February 2019 / Published: 18 February 2019
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Abstract
A bio-orthogonal and unnatural substance, such as an unnatural amino acid (Uaa), is an ideal regulator to control target gene expression in a synthetic gene circuit. Genetic code expansion technology has achieved Uaa incorporation into ribosomal synthesized proteins in vivo at specific sites [...] Read more.
A bio-orthogonal and unnatural substance, such as an unnatural amino acid (Uaa), is an ideal regulator to control target gene expression in a synthetic gene circuit. Genetic code expansion technology has achieved Uaa incorporation into ribosomal synthesized proteins in vivo at specific sites designated by UAG stop codons. This site-specific Uaa incorporation can be used as a controller of target gene expression at the translational level by conditional read-through of internal UAG stop codons. Recent advances in optimization of site-specific Uaa incorporation for translational regulation have enabled more precise control over a wide range of novel important applications, such as Uaa-auxotrophy-based biological containment, live-attenuated vaccine, and high-yield zero-leakage expression systems, in which Uaa translational control is exclusively used as an essential genetic element. This review summarizes the history and recent advance of the translational control by conditional stop codon read-through, especially focusing on the methods using the site-specific Uaa incorporation. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessReview
Engineering Translation Components Improve Incorporation of Exotic Amino Acids
Int. J. Mol. Sci. 2019, 20(3), 522; https://doi.org/10.3390/ijms20030522
Received: 2 January 2019 / Revised: 19 January 2019 / Accepted: 21 January 2019 / Published: 26 January 2019
PDF Full-text (1796 KB) | HTML Full-text | XML Full-text
Abstract
Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. [...] Read more.
Methods of genetic code manipulation, such as nonsense codon suppression and genetic code reprogramming, have enabled the incorporation of various nonproteinogenic amino acids into the peptide nascent chain. However, the incorporation efficiency of such amino acids largely varies depending on their structural characteristics. For instance, l-α-amino acids with artificial, bulky side chains are poorer substrates for ribosomal incorporation into the nascent peptide chain, mainly owing to the lower affinity of their aminoacyl-tRNA toward elongation factor-thermo unstable (EF-Tu). Phosphorylated Ser and Tyr are also poorer substrates for the same reason; engineering EF-Tu has turned out to be effective in improving their incorporation efficiencies. On the other hand, exotic amino acids such as d-amino acids and β-amino acids are even poorer substrates owing to their low affinity to EF-Tu and poor compatibility to the ribosome active site. Moreover, their consecutive incorporation is extremely difficult. To solve these problems, the engineering of ribosomes and tRNAs has been executed, leading to successful but limited improvement of their incorporation efficiency. In this review, we comprehensively summarize recent attempts to engineer the translation systems, resulting in a significant improvement of the incorporation of exotic amino acids. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Open AccessReview
Expanding the Genetic Code for Site-Directed Spin-Labeling
Int. J. Mol. Sci. 2019, 20(2), 373; https://doi.org/10.3390/ijms20020373
Received: 19 December 2018 / Revised: 10 January 2019 / Accepted: 15 January 2019 / Published: 16 January 2019
Cited by 1 | PDF Full-text (1998 KB) | HTML Full-text | XML Full-text
Abstract
Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSL–EPR experiments crucially depends on the employed labeling strategy, with key [...] Read more.
Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy enables studies of the structure, dynamics, and interactions of proteins in the noncrystalline state. The scope and analytical value of SDSL–EPR experiments crucially depends on the employed labeling strategy, with key aspects being labeling chemoselectivity and biocompatibility, as well as stability and spectroscopic properties of the resulting label. The use of genetically encoded noncanonical amino acids (ncAA) is an emerging strategy for SDSL that holds great promise for providing excellent chemoselectivity and potential for experiments in complex biological environments such as living cells. We here give a focused overview of recent advancements in this field and discuss their potentials and challenges for advancing SDSL–EPR studies. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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Other

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Open AccessConference Report
Expanding Genetic Code Expansion through Resource Facilities, Workshops, and Conferences
Int. J. Mol. Sci. 2019, 20(9), 2103; https://doi.org/10.3390/ijms20092103
Received: 26 March 2019 / Revised: 19 April 2019 / Accepted: 22 April 2019 / Published: 29 April 2019
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Abstract
Genetic Code Expansion (GCE) enables the encoding of amino acids with diverse chemical properties. This approach has tremendous potential to advance biological discoveries in basic research, medical, and industrial settings. Given the multiple technical approaches and the associated research activities used to achieve [...] Read more.
Genetic Code Expansion (GCE) enables the encoding of amino acids with diverse chemical properties. This approach has tremendous potential to advance biological discoveries in basic research, medical, and industrial settings. Given the multiple technical approaches and the associated research activities used to achieve GCE, herein we have taken the opportunity to describe ongoing out-reach efforts in the GCE community. These include Resource Facilities that nucleate expertise and reagents within a specific GCE discipline, hands-on Workshops to provide GCE training, and GCE Conferences which bring the community together in a collegial setting. The overall goal of these activities is to accelerate the integration of GCE approaches into more research settings and to facilitate solutions to common technical hurdles. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code)
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