Establishing the fundamental rules for genetic code expansion.
Ontology highlight
ABSTRACT:
SUBMITTER: Sinha S
PROVIDER: S-EPMC10460129 | biostudies-literature | 2023 Jul
REPOSITORIES: biostudies-literature
Publications
Establishing the fundamental rules for genetic code expansion.
Sinha Souvik S Ahsan Mohd M Palermo Giulia G
Nature chemistry 20230701 7
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Project description:Expanding the genetic code to enable the incorporation of unnatural amino acids into proteins in biological systems provides a powerful tool for studying protein structure and function. While this technology has been mostly developed and applied in bacterial and mammalian cells, it recently expanded into animals, including worms, fruit flies, zebrafish, and mice. In this review, we highlight recent advances toward the methodology development of genetic code expansion in animal model organisms. We further illustrate the applications, including proteomic labeling in fruit flies and mice and optical control of protein function in mice and zebrafish. We summarize the challenges of unnatural amino acid mutagenesis in animals and the promising directions toward broad application of this emerging technology.
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2024-12-15 | PXD052776 | Pride
Project description:Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T-REx or Tet-On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T-REx system enables simultaneous control of tRNA and RS expression.
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Project description:The advancement of genetic code expansion (GCE) technology is attributed to the establishment of specific aminoacyl-tRNA synthetase/tRNA pairs. While earlier improvements mainly focused on aminoacyl-tRNA synthetases, recent studies have highlighted the importance of optimizing tRNA sequences to enhance both unnatural amino acid incorporation efficiency and orthogonality. Given the crucial role of tRNAs in the translation process and their substantial impact on overall GCE efficiency, ongoing efforts are dedicated to the development of tRNA engineering techniques. This review explores diverse tRNA engineering approaches and provides illustrative examples in the context of GCE, offering insights into the user-friendly implementation of GCE technology.
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Project description:By providing long-term protection against infectious diseases, vaccinations have significantly reduced death and morbidity worldwide. In the 21st century, (bio)technological advances have paved the way for developing prophylactic vaccines that are safer and more effective as well as enabling the use of vaccines as therapeutics to treat human diseases. Here, we provide a focused review of the utility of genetic code expansion as an emerging tool for the development of vaccines. Specifically, we discuss how the incorporation of immunogenic noncanonical amino acids can aid in eliciting immune responses against adverse self-proteins and highlight the potential of an expanded genetic code for the construction of replication-incompetent viruses. We close the review by discussing the future prospects and remaining challenges for the application of these approaches in the development of both prophylactic and therapeutic vaccines in the near future.
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Project description:Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.
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Project description:Multiple applications of genome editing by CRISPR-Cas9 necessitate stringent regulation and Cas9 variants have accordingly been generated whose activity responds to small ligands, temperature or light. However, these approaches are often impracticable, for example in clinical therapeutic genome editing in situ or gene drives in which environmentally-compatible control is paramount. With this in mind, we have developed heritable Cas9-mediated mammalian genome editing that is acutely controlled by the cheap lysine derivative, Lys(Boc) (BOC). Genetic code expansion permitted non-physiological BOC incorporation such that Cas9 (Cas9BOC) was expressed in a full-length, active form in cultured somatic cells only after BOC exposure. Stringently BOC-dependent, heritable editing of transgenic and native genomic loci occurred when Cas9BOC was expressed at the onset of mouse embryonic development from cRNA or Cas9BOC transgenic females. The tightly controlled Cas9 editing system reported here promises to have broad applications and is a first step towards purposed, spatiotemporal gene drive regulation over large geographical ranges.
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Project description:Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.
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Project description:Escherichia coli has been considered as the most used model bacteria in the majority of studies for several decades. However, a new, faster chassis for synthetic biology is emerging in the form of the fast-growing gram-negative bacterium Vibrio natriegens. Different methodologies, well established in E. coli, are currently being adapted for V. natriegens in the hope to enable a much faster platform for general molecular biology studies. Amongst the vast technologies available for E. coli, genetic code expansion, the incorporation of unnatural amino acids into proteins, serves as a robust tool for protein engineering and biorthogonal modifications. Here we designed and adapted the genetic code expansion methodology for V. natriegens and demonstrate an unnatural amino acid incorporation into a protein for the first time in this organism.
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Project description:Protein post-translational modification regulates near every aspect of cellular processes in eukaryotes. However, the identification of new protein post-translational modification is very challenging. Here, using genetically encoded unnatural amino acids as chemical probes, we report the identification and validation of a previously unreported form of protein post-translational modification, aminoacylated lysine ubiquitination, in which the modification occurs on the α-amine group of aminoacylated lysine. We identify more than two thousands of unique aminoacylated lysine ubiquitination sites on all 20 aminoacylated lysines in two human cell lines. The aminoacylated lysine ubiquitination modifications can mediate rapid protein degradation, complementing the canonical lysine ubiquitination-mediated proteome degradation. Furthermore, we demonstrate that the ubiquitin conjugating enzyme UBE2W acts as a writer of aminoacylated lysine ubiquitination and facilitates the ubiquitination of internal α-amine groups on proteins using an unexpected enzymatic activity. More broadly, the discovery and validation of aminoacylated lysine ubiquitination paves the way for the identification and verification of new protein post-translation modifications with the genetic code expansion strategy.
2022-07-20 | PXD035166 |