Genetic Code Expansion: A Brief History and Perspective

Shandell, Mia A.; Tan, Zhongping; Cornish, Virginia W.

doi:10.1021/acs.biochem.1c00286

Cited by 103 publications

(84 citation statements)

References 116 publications

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“…Conditional protein modifications are fundamental in the life sciences. Genetic code expansion connects the versatility of chemical synthesis to protein expression in living systems. − Protein modifications can be inserted site-specifically via natural translation machineries . By reprogramming the genetic code, non-canonical amino acids bearing a modification of choice are used in ribosomal polypeptide synthesis. − Non-canonical amino acids have many applications in protein engineering, as they can be equipped with isotopes for structural studies, − photoreactive groups and post-translational modifications for functional studies, − reactive groups for bio-orthogonal coupling, ,,− photocross-linkers, ,,− infrared-active probes to follow conformational dynamics, ,− fluorescent dyes for imaging, − biotin analogues for high-affinity interactions with streptavidin, and stable phosphotyrosine analogues for analysis of signal transduction. , Site-specific incorporation is achieved by suppressing a stop codon with an additional aminoacyl-tRNA-synthetase (aaRS)/tRNA pair. ,,, In bacterial or mammalian cells, the rarest amber codon (TAG) is most often used to minimize suppression throughout the proteome. ,,,, However, the underlying processes of the genetic code expansion are complex and hardly understood. ,…”

Section: Introductionmentioning

confidence: 99%

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

et al. 2022

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Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

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Section: Introductionmentioning

confidence: 99%

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

et al. 2022

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“…A complementary method to AP-MS and PL for analyzing PPIs involves covalent cross-linking (Liu et al, 2015;Wang, 2017;Nguyen et al, 2018;Yu and Huang, 2018). Genetic code expansion by amber codon suppression has enabled sitespecific incorporation of non-canonical amino acids (ncAAs) into proteins (Wang et al, 2001;Wang and Schultz, 2004;Wang et al, 2006;de la Torre and Chin, 2021;Shandell et al, 2021). Many ncAAs, including photo-activated ncAAs (Chin et al, 2002a;Chin et al, 2002b;Zhang et al, 2011;Lin et al, 2014;Yang et al, 2016) and those with fine-tuned bio-reactivity to capture protein binders (Xiang et al, 2013;Chen X.-H. et al, 2014;Furman et al, 2014;Xiang et al, 2014;Xuan et al, 2016;Cigler et al, 2017;Wang, 2017;Nguyen et al, 2018;Shang et al, 2018;Wang et al, 2018;Yang et al, 2019;Liu et al, 2020;Liu J. et al, 2021), has been successfully incorporated into proteins.…”

Section: Introductionmentioning

confidence: 99%

Uncover New Reactivity of Genetically Encoded Alkyl Bromide Non-Canonical Amino Acids

et al. 2022

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Genetically encoded non-canonical amino acids (ncAAs) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These ncAAs, including a series of alkyl bromide-based ncAAs, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based ncAA, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interaction interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.

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“…Some of GCE’s applications have been to probe the structure and function of proteins, alter their redox potential, introduce fluorophores, infrared, and spin label probes, encode post-translational modifications, and create sites for site-specific bioconjugation. Several recent reviews of how GCE has been used to modify proteins expressed in bacteria, yeast, and mammalian cells can be found elsewhere ( Young and Schultz, 2018 ; Chung et al, 2020 ; Nikić-Spiegel, 2020 ; Manandhar et al, 2021 ; Ros et al, 2021 ; Shandell et al, 2021 ; Sanders et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Expanding the chemical diversity of M13 bacteriophage

et al. 2022

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Bacteriophage M13 virions are very stable nanoparticles that can be modified by chemical and genetic methods. The capsid proteins can be functionalized in a variety of chemical reactions without loss of particle integrity. In addition, Genetic Code Expansion (GCE) permits the introduction of non-canonical amino acids (ncAAs) into displayed peptides and proteins. The incorporation of ncAAs into phage libraries has led to the discovery of high-affinity binders with low nanomolar dissociation constant (KD) values that can potentially serve as inhibitors. This article reviews how bioconjugation and the incorporation of ncAAs during translation have expanded the chemistry of peptides and proteins displayed by M13 virions for a variety of purposes.

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Genetic Code Expansion: A Brief History and Perspective

Cited by 103 publications

References 116 publications

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

Uncover New Reactivity of Genetically Encoded Alkyl Bromide Non-Canonical Amino Acids

Expanding the chemical diversity of M13 bacteriophage

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