Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes

Italia, James S.; Latour, Christopher; Wrobel, Chester J J; Chatterjee, Abhishek

doi:10.1016/j.chembiol.2018.07.002

Cited by 50 publications

(92 citation statements)

References 50 publications

(122 reference statements)

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“…The orthogonality feature of these pairs determines the disadvantage that two distinct platforms need to be developed for applications of one single ncAA in both bacterial and eukaryotic cells. Italia, J. S. et al recently reported a novel strategy to expand the genetic code of E. coli by replacing its endogenous pair with an eukaryotic-archaeal counterpart, achieving a general way to develop new aaRS/tRNA pairs for both E. coli and eukaryotes [ 34 , 41 ].…”

Section: Introductionmentioning

confidence: 99%

Therapeutic applications of genetic code expansion

Huang

Liu

2018

Synthetic and Systems Biotechnology

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In nature, a limited, conservative set of amino acids are utilized to synthesize proteins. Genetic code expansion technique reassigns codons and incorporates noncanonical amino acids (ncAAs) through orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The past decade has witnessed the rapid growth in diversity and scope for therapeutic applications of this technology. Here, we provided an update on the recent progress using genetic code expansion in the following areas: antibody-drug conjugates (ADCs), bispecific antibodies (BsAb), immunotherapies, long-lasting protein therapeutics, biosynthesized peptides, engineered viruses and cells, as well as other therapeutic related applications, where the technique was used to elucidate the mechanisms of biotherapeutics and drug targets.

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Section: Introductionmentioning

confidence: 99%

Therapeutic applications of genetic code expansion

Huang

Liu

2018

Synthetic and Systems Biotechnology

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“…M. alvus TyrRS and Y072/Y005M and the pairs of Cs HisRS variants and αH005-αψHis-4 variants may be orthogonal in E. coli and might be useful for incorporating non-canonical amino acids [ 17 , 58 , 60 ]. Alternatively, some of the endogenous tRNAs or tRNA-aaRS pairs might be replaceable with allo-tRNAs or orthogonal pairs composed of allo-tRNAs [ 60 , 63 , 64 , 65 ]. For reprogramming the genetic code, the allo-tRNA sequestration system may be useful for use in vivo and in vitro [ 66 ] and for the purification and elimination of allo-tRNA molecules from tRNA mixtures [ 66 , 67 , 68 , 69 ].…”

Section: Discussionmentioning

confidence: 99%

Rational Design of Aptamer-Tagged tRNAs

Mukai

2020

IJMS

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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.

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“…3B and C). The first co-evolved tRNA Tyr /TyrRS pairs suffered from limited orthogonality (Wang et al 2001;Chin et al 2003), but further developments now allow archaeal or modified bacterial tRNA/aaRS pairs to be employed for both rapid selection in bacteria and application of the pairs in mammalian cells (Mukai et al 2008;Neumann et al 2008;Yanagisawa et al 2008;Chen et al 2009;Italia et al 2018Italia et al , 2020. An overview of the most common tRNA/aaRS pairs is provided in Chin (2017), while the selection process for synthetases is reviewed in Davis & Chin (2012) and Chin (2014).…”

Section: Nonsense Suppression With Co-evolved Trna and Aminoacyl-trnamentioning

confidence: 99%

The current chemical biology tool box for studying ion channels

Braun

Sheikh

Pless

2020

The Journal of Physiology

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Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be Nina Braun studied biochemistry at the University of Bayreuth and Stockholm University before discovering her interest in ion channels and non-canonical amino acids while working on her Masters thesis with Stephan A. Pless at the University of Copenhagen. She completed her PhD in the Pless lab and is currently working on high-throughput assessment of non-canonical amino acid incorporation using photocrosslinkers in acid-sensing ion channels, with the goal of studying protein-protein interactions. Zeshan P. Sheikh completed his MSc degree at the University of Copenhagen, where he discovered his passion for ion channels, mainly focusing on recombinant expression and purification. He embarked on a PhD in the Pless lab pursuing his interest in ion channel biophysics. Here, he gained particular interest in the application of chemical biological techniques for modifying ion channels. His current research priority is applying split inteins to modify acid-sensing ion channels to gain insights into their structural dynamics.

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Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes

Cited by 50 publications

References 50 publications

Therapeutic applications of genetic code expansion

Therapeutic applications of genetic code expansion

Rational Design of Aptamer-Tagged tRNAs

The current chemical biology tool box for studying ion channels

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