An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells

Osgood, Arianna O.; Zheng, Yunan; Roy, Soumya Jyoti Singha; Loynd, Conor; Jewel, Delilah; Chatterjee, Abhishek

doi:10.1101/2022.08.02.502539

Cited by 4 publications

(6 citation statements)

References 40 publications

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“…Our study highlights opportunities and limitations of dual suppression systems, requiring two selective, efficient and mutual orthogonal incorporation machineries. The relatively lower efficiency of ochre and opal stop codon suppression limits overall yield of dually suppressed protein (Zheng et al 2017; Meineke et al 2018, 2020; Osgood et al 2022). We chose AzF for encoding the CuAAC handle because of its efficient incorporation (Figure 2A, S2B), but this likely limited the fluorescent labeling efficiency on live cells.…”

Section: Discussionmentioning

confidence: 99%

“…Most commonly the amber (TAG) stop codon is repurposed, using an engineered tRNA/aminoacyl-tRNA synthetase (aaRS) pair to deliver an ncAA-charged tRNA to the ribosome. The amber codon is the least abundant (23% in the human genome) and most efficiently suppressed stop codon, but the other two stop codons UUA (ochre) and UGA (opal) have also been exploited for ncAA incorporation in mammalian cells and enabled dual ncAA incorporation via dual suppression (Xiao et al 2013; Zheng et al 2017) (Meineke et al 2018, 2020; Serfling et al 2018; Beranek et al 2019; Osgood et al 2022). Quadruplet codon suppression has been proven possible in worms and mammalian cells as well (Niu et al 2013; Xi et al 2022).…”

Section: Introductionmentioning

confidence: 99%

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Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Meineke

Caridha

Block

et al. 2023

Preprint

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Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Suppressor tRNAs are aminoacylated with noncanonical amino acids (ncAAs) by dedicated aminoacyl-tRNA synthetases (aaRS) and direct ncAA incorporation site-specifically during translation. These pairs of tRNA/aaRS must be orthogonal to the host's tRNAs, aaRS and natural amino acids. Pyrrolysyl-tRNA (PylT)/PylRS pairs from methanogenic archaea, as well as engineered tRNA/aaRS pairs derived from bacteria, are used for genetic code expansion in mammalian cells. Amber suppression is routinely achieved by transient introduction of the components leading to short-term and heterogeneous expression. Here, we demonstrate that stable integration of tRNA/aaRS genes allows for efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We extend a general plasmid design and PiggyBac (PB) integration strategy developed for the Methanosarcina mazei PylT/PylRS pair to genomic integration of two tRNA/aaRS pairs of bacterial origin. We further explore suppression of ochre and opal stop codons and parallel incorporation of two distinct ncAAs, both accessible for click chemistry, by dual suppression in stable cell lines. Clonal selection allows for isolation of cells with high dual suppression efficiency and dual site-specific fluorescent labeling of a cell surface receptor using bioorthogonal click chemistries on live mammalian cells.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Meineke

Caridha

Block

et al. 2023

Preprint

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“…While a portion of this challenge can be mitigated by using high ncAA concentrations during induction of protein synthesis, the discovery of additional high-performing aaRS variants that discriminate against the canonical amino acids will be important going forward. Dual ncAA incorporation in yeast will certainly benefit from ongoing efforts to expand the availability and performance of OTSs to access a broader palette of genetically encodable ncAAs with useful chemistries (Dunkelmann et al, 2020; Grasso et al, 2022; He et al, 2020; Italia et al, 2017; Osgood et al, 2022; Stieglitz and Van Deventer, 2021; Tan et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

“…Parallel efforts from our lab have led to the discovery of a broad range of E. coli tyrosyl-tRNA synthetase ( Ec TyrRS) and E. coli Leucyl-tRNA synthetase ( Ec LeuRS) variants with a variety of ncAA incorporation properties, including new-to-yeast ncAA incorporation and improved selectivity and activity for known ncAAs (Stieglitz and Van Deventer, 2022). In addition, work by Chatterjee and coworkers in engineered E. coli strains has led to the identification of additional Ec aaRS variants derived from Ec TyRS and Ec TrpRS with unique substrate preferences for use in mammalian cells (Grasso et al, 2022; Osgood et al, 2022). While not yet evaluated in yeast, such Ec aaRS variants have the potential to further broaden the range of OTSs available for use in this organism.…”

Section: Introductionmentioning

confidence: 99%

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

Lahiri

Deventer

2022

Preprint

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Proteins containing noncanonical amino acids (ncAAs) provide opportunities for dissecting fundamental biological processes and engineering protein therapeutics with diverse chemistries. Incorporation of more than one ncAA into a single construct can endow a protein with multiple useful features, such as unique molecular recognition and covalent crosslinking. Herein, for the first time, we encode two chemically distinct ncAAs into proteins prepared in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems (OTSs): E. coli tyrosyl-tRNA synthetase/tRNATyr, E. coli leucyl-tRNA synthetase/tRNALeu, and M. alvus pyrrolysyl-tRNA synthetase/tRNAPyl. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Further, we found that multiple factors impacted TGA readthrough efficiency in a manner analogous to TAG readthrough, including the local nucleotide context surrounding stop codons and deletion of selected genes from the yeast genome. In contrast, the identity of the suppressor tRNA affected TGA readthrough efficiency quite differently than TAG readthrough efficiency. Altogether, these observations facilitated the systematic investigation of dual ncAA incorporation in response to TAG and TGA codons within both intracellular and yeast-displayed protein constructs. Employing a proficient combination of OTSs, pairs of ncAAs could be encoded within both protein formats with efficiencies up to 6% of wildtype protein controls. Moreover, exploration of dual ncAA incorporation in yeast display format aided in demonstrating important applications of doubly-substituted proteins. First, dual ncAA incorporation within a simple synthetic antibody construct resulted in retention of antigen binding functionality, indicating that the doubly-substituted proteins can retain their basic functions. Second, presentation of reactive alkyne and azide functionalities within yeast-displayed constructs enabled sequential installation of two distinct chemical probes using bioorthogonal reactions--strain-promoted- and copper-catalyzed azide-alkyne cycloadditions (SPAAC and CuAAC)--on the yeast surface. This confirmed the ability to chemoselectively modify yeast-displayed proteins at two distinct sites. Furthermore, by utilizing a soluble form of a doubly substituted construct, we validated the feasibility to selectively double label proteins in solution in a single pot reaction. Overall, our work facilitates the addition of a 22nd amino acid to the yeast genetic code and provides an important toolkit that expands the scope of applications of ncAAs for basic biological research and drug discovery in yeast.

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“…38 Similar efforts by Chatterjee and co-workers in engineered E. coli strains have led to the identification of additional EcaaRS variants derived from EcTyRS and EcTrpRS with unique substrate preferences for use in mammalian cells. 33,39 While not yet evaluated in yeast, such EcaaRS variants have the potential to further broaden the range of OTSs available for use in this organism. Beyond E. coli-derived OTSs, translation machinery derived from archaeal Pyrrolysyl-tRNA synthetases (PylRS)-tRNA pairs continues to mature.…”

Section: ■ Introductionmentioning

confidence: 99%

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

et al. 2023

View full text Add to dashboard Cite

Incorporation of more than one noncanonical amino acid (ncAA) within a single protein endows the resulting construct with multiple useful features such as augmented molecular recognition or covalent cross-linking capabilities. Herein, for the first time, we demonstrate the incorporation of two chemically distinct ncAAs into proteins biosynthesized in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Readthrough efficiency at TGA was modulated by factors including the local nucleotide environment, gene deletions related to the translation process, and the identity of the suppressor tRNA. These observations facilitated systematic investigation of dual ncAA incorporation in both intracellular and yeast-displayed protein constructs, where we observed efficiencies up to 6% of wild-type protein controls. The successful display of doubly substituted proteins enabled the exploration of two critical applications on the yeast surface(A) antigen binding functionality and (B) chemoselective modification with two distinct chemical probes through sequential application of two bioorthogonal click chemistry reactions. Lastly, by utilizing a soluble form of a doubly substituted construct, we validated the dual incorporation system using mass spectrometry and demonstrated the feasibility of conducting selective labeling of the two ncAAs sequentially using a “single-pot” approach. Overall, our work facilitates the addition of a 22nd amino acid to the genetic code of yeast and expands the scope of applications of ncAAs for basic biological research and drug discovery.

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An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells

Cited by 4 publications

References 40 publications

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

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