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Expanding and Reprogramming the Genetic Code 2.0
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Expanding and Reprogramming the Genetic Code 2.0

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 (28 February 2022) | Viewed by 27003

Special Issue Editor

Dr. Kensaku Sakamoto

E-Mail Website
Guest Editor
Riken, Division of Structural and Synthetic Biology, Wako, Japan
Interests: genetic code expansion; codon reassignment; tRNA; aminoacyl-tRNA synthetase
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

This Special Issue is a continuation of our previous Special Issue “Expanding and Reprogramming the Genetic Code”.

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

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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

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Related Special Issue

Published Papers (6 papers)

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Research

11 pages, 1687 KiB  
Article
Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography
by Toshiaki Hosaka, Kazushige Katsura, Yoshiko Ishizuka-Katsura, Kazuharu Hanada, Kaori Ito, Yuri Tomabechi, Mio Inoue, Ryogo Akasaka, Chie Takemoto and Mikako Shirouzu
Int. J. Mol. Sci. 2022, 23(18), 10399; https://doi.org/10.3390/ijms231810399 - 8 Sep 2022
Cited by 2 | Viewed by 2026
Abstract
Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its [...] Read more.
Genetically encoded caged amino acids can be used to control the dynamics of protein activities and cellular localization in response to external cues. In the present study, we revealed the structural basis for the recognition of O-(2-nitrobenzyl)-L-tyrosine (oNBTyr) by its specific variant of Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (oNBTyrRS), and then demonstrated its potential availability for time-resolved X-ray crystallography. The substrate-bound crystal structure of oNBTyrRS at a 2.79 Å resolution indicated that the replacement of tyrosine and leucine at positions 32 and 65 by glycine (Tyr32Gly and Leu65Gly, respectively) and Asp158Ser created sufficient space for entry of the bulky substitute into the amino acid binding pocket, while Glu in place of Leu162 formed a hydrogen bond with the nitro moiety of oNBTyr. We also produced an oNBTyr-containing lysozyme through a cell-free protein synthesis system derived from the Escherichia coli B95. ΔA strain with the UAG codon reassigned to the nonnatural amino acid. Another crystallographic study of the caged protein showed that the site-specifically incorporated oNBTyr was degraded to tyrosine by light irradiation of the crystals. Thus, cell-free protein synthesis of caged proteins with oNBTyr could facilitate time-resolved structural analysis of proteins, including medically important membrane proteins. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code 2.0)
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8 pages, 964 KiB  
Communication
Plasmid Curing and Exchange Using a Novel Counter-Selectable Marker Based on Unnatural Amino Acid Incorporation at a Sense Codon
by Yusuke Kato
Int. J. Mol. Sci. 2021, 22(21), 11482; https://doi.org/10.3390/ijms222111482 - 25 Oct 2021
Cited by 2 | Viewed by 4526
Abstract
A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and [...] Read more.
A protocol was designed for plasmid curing using a novel counter-selectable marker, named pylSZK-pylT, in Escherichia coli. The pylSZK-pylT marker consists of the archaeal pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA (tRNApyl) with modification, and incorporates an unnatural amino acid (Uaa), Nε-benzyloxycarbonyl-l-lysine (ZK), at a sense codon in ribosomally synthesized proteins, resulting in bacterial growth inhibition or killing. Plasmid curing is performed by exerting toxicity on pylSZK-pylT located on the target plasmid, and selecting only proliferative bacteria. All tested bacteria obtained using this protocol had lost the target plasmid (64/64), suggesting that plasmid curing was successful. Next, we attempted to exchange plasmids with the identical replication origin and an antibiotic resistance gene without plasmid curing using a modified protocol, assuming substitution of plasmids complementing genomic essential genes. All randomly selected bacteria after screening had only the substitute plasmid and no target plasmid (25/25), suggesting that plasmid exchange was also accomplished. Counter-selectable markers based on PylRS-tRNApyl, such as pylSZK-pylT, may be scalable in application due to their independence from the host genotype, applicability to a wide range of species, and high tunability due to the freedom of choice of target codons and Uaa’s to be incorporated. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code 2.0)
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17 pages, 1850 KiB  
Article
Engineering Pyrrolysyl-tRNA Synthetase for the Incorporation of Non-Canonical Amino Acids with Smaller Side Chains
by Nikolaj G. Koch, Peter Goettig, Juri Rappsilber and Nediljko Budisa
Int. J. Mol. Sci. 2021, 22(20), 11194; https://doi.org/10.3390/ijms222011194 - 17 Oct 2021
Cited by 15 | Viewed by 8593
Abstract
Site-specific incorporation of non-canonical amino acids (ncAAs) into proteins has emerged as a universal tool for systems bioengineering at the interface of chemistry, biology, and technology. The diversification of the repertoire of the genetic code has been achieved for amino acids with long [...] Read more.
Site-specific incorporation of non-canonical amino acids (ncAAs) into proteins has emerged as a universal tool for systems bioengineering at the interface of chemistry, biology, and technology. The diversification of the repertoire of the genetic code has been achieved for amino acids with long and/or bulky side chains equipped with various bioorthogonal tags and useful spectral probes. Although ncAAs with relatively small side chains and similar properties are of great interest to biophysics, cell biology, and biomaterial science, they can rarely be incorporated into proteins. To address this gap, we report the engineering of PylRS variants capable of incorporating an entire library of aliphatic “small-tag” ncAAs. In particular, we performed mutational studies of a specific PylRS, designed to incorporate the shortest non-bulky ncAA (S-allyl-l-cysteine) possible to date and based on this knowledge incorporated aliphatic ncAA derivatives. In this way, we have not only increased the number of translationally active “small-tag” ncAAs, but also determined key residues responsible for maintaining orthogonality, while engineering the PylRS for these interesting substrates. Based on the known plasticity of PylRS toward different substrates, our approach further expands the reassignment capacities of this enzyme toward aliphatic amino acids with smaller side chains endowed with valuable functionalities. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code 2.0)
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14 pages, 1656 KiB  
Communication
Method for Rapid Analysis of Mutant RNA Polymerase Activity on Templates Containing Unnatural Nucleotides
by Tatiana Egorova, Ekaterina Shuvalova, Sabina Mukba, Alexey Shuvalov, Peter Kolosov and Elena Alkalaeva
Int. J. Mol. Sci. 2021, 22(10), 5186; https://doi.org/10.3390/ijms22105186 - 14 May 2021
Cited by 3 | Viewed by 2869
Abstract
Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing [...] Read more.
Pairs of unnatural nucleotides are used to expand the genetic code and create artificial DNA or RNA templates. In general, an approach is used to engineer orthogonal systems capable of reading codons comprising artificial nucleotides; however, DNA and RNA polymerases capable of recognizing unnatural nucleotides are required for amplification and transcription of templates. Under favorable conditions, in the presence of modified nucleotide triphosphates, DNA polymerases are able to synthesize unnatural DNA with high efficiency; however, the currently available RNA polymerases reveal high specificity to the natural nucleotides and may not easily recognize the unnatural nucleotides. Due to the absence of simple and rapid methods for testing the activity of mutant RNA polymerases, the development of RNA polymerase recognizing unnatural nucleotides is limited. To fill this gap, we developed a method for rapid analysis of mutant RNA polymerase activity on templates containing unnatural nucleotides. Herein, we optimized a coupled cell-free translation system and tested the ability of three unnatural nucleotides to be transcribed by different T7 RNA polymerase mutants, by demonstrating high sensitivity and simplicity of the developed method. This approach can be applied to various unnatural nucleotides and can be simultaneously scaled up to determine the activity of numerous polymerases on different templates. Due to the simplicity and small amounts of material required, the developed cell-free system provides a highly scalable and versatile tool to study RNA polymerase activity. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code 2.0)
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18 pages, 1361 KiB  
Article
Directed Evolution of the Methanosarcina barkeri Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential
by David G. Schwark, Margaret A. Schmitt and John D. Fisk
Int. J. Mol. Sci. 2021, 22(2), 895; https://doi.org/10.3390/ijms22020895 - 18 Jan 2021
Cited by 13 | Viewed by 3568
Abstract
Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber [...] Read more.
Genetic code expansion has largely focused on the reassignment of amber stop codons to insert single copies of non-canonical amino acids (ncAAs) into proteins. Increasing effort has been directed at employing the set of aminoacyl tRNA synthetase (aaRS) variants previously evolved for amber suppression to incorporate multiple copies of ncAAs in response to sense codons in Escherichia coli. Predicting which sense codons are most amenable to reassignment and which orthogonal translation machinery is best suited to each codon is challenging. This manuscript describes the directed evolution of a new, highly efficient variant of the Methanosarcina barkeri pyrrolysyl orthogonal tRNA/aaRS pair that activates and incorporates tyrosine. The evolved M. barkeri tRNA/aaRS pair reprograms the amber stop codon with 98.1 ± 3.6% efficiency in E. coli DH10B, rivaling the efficiency of the wild-type tyrosine-incorporating Methanocaldococcus jannaschii orthogonal pair. The new orthogonal pair is deployed for the rapid evaluation of sense codon reassignment potential using our previously developed fluorescence-based screen. Measurements of sense codon reassignment efficiencies with the evolved M. barkeri machinery are compared with related measurements employing the M. jannaschii orthogonal pair system. Importantly, we observe different patterns of sense codon reassignment efficiency for the M. jannaschii tyrosyl and M. barkeri pyrrolysyl systems, suggesting that particular codons will be better suited to reassignment by different orthogonal pairs. A broad evaluation of sense codon reassignment efficiencies to tyrosine with the M. barkeri system will highlight the most promising positions at which the M. barkeri orthogonal pair may infiltrate the E. coli genetic code. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code 2.0)
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18 pages, 3922 KiB  
Article
Rational Design of Aptamer-Tagged tRNAs
by Takahito Mukai
Int. J. Mol. Sci. 2020, 21(20), 7793; https://doi.org/10.3390/ijms21207793 - 21 Oct 2020
Cited by 2 | Viewed by 3893
Abstract
Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I [...] Read more.
Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code. Full article
(This article belongs to the Special Issue Expanding and Reprogramming the Genetic Code 2.0)
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