Browsing by Author "Tharp, Jeffery M."

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    A Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2
    (American Chemical Society, 2022) Hampton, J. Trae; Lalonde, Tyler J.; Tharp, Jeffery M.; Kurra, Yadagiri; Alugubelli, Yugendar R.; Roundy, Christopher M.; Hamer, Gabriel L.; Xu, Shiqing; Liu, Wenshe Ray; Biochemistry and Molecular Biology, School of Medicine
    Using the regioselective cyanobenzothiazole condensation reaction with an N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and ACE2, the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2 showed that one macrocyclic peptide is highly potent against viral reproduction in Vero E6 cells with an EC50 value of 3.1 μM. The AlphaLISA-detected IC50 value for this macrocyclic peptide was 0.3 μM. The current study demonstrates that two kinetically-controlled reactions toward N-terminal and internal cysteines, respectively, are highly effective in the construction of phage-displayed macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike epitopes is a promising methodology in the identification of peptidyl antivirals.
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    Ancestral archaea expanded the genetic code with pyrrolysine
    (Elsevier, 2022) Guo, Li-Tao; Amikura, Kazuaki; Jiang, Han-Kai; Mukai, Takahito; Fu, Xian; Wang, Yane-Shih; O’Donoghue, Patrick; Söll, Dieter; Tharp, Jeffery M.; Biochemistry and Molecular Biology, School of Medicine
    The pyrrolysyl-tRNA synthetase (PylRS) facilitates the cotranslational installation of the 22nd amino acid pyrrolysine. Owing to its tolerance for diverse amino acid substrates, and its orthogonality in multiple organisms, PylRS has emerged as a major route to install noncanonical amino acids into proteins in living cells. Recently, a novel class of PylRS enzymes was identified in a subset of methanogenic archaea. Enzymes within this class (ΔPylSn) lack the N-terminal tRNA-binding domain that is widely conserved amongst PylRS enzymes, yet remain active and orthogonal in bacteria and eukaryotes. In this study, we use biochemical and in vivo UAG-readthrough assays to characterize the aminoacylation efficiency and substrate spectrum of a ΔPylSn class PylRS from the archaeon Candidatus Methanomethylophilus alvus. We show that, compared with the full-length enzyme from Methanosarcina mazei, the Ca. M. alvus PylRS displays reduced aminoacylation efficiency but an expanded amino acid substrate spectrum. To gain insight into the evolution of ΔPylSn enzymes, we performed molecular phylogeny using 156 PylRS and 105 pyrrolysine tRNA (tRNAPyl) sequences from diverse archaea and bacteria. This analysis suggests that the PylRS•tRNAPyl pair diverged before the evolution of the three domains of life, placing an early limit on the evolution of the Pyl-decoding trait. Furthermore, our results document the coevolutionary history of PylRS and tRNAPyl and reveal the emergence of tRNAPyl sequences with unique A73 and U73 discriminator bases. The orthogonality of these tRNAPyl species with the more common G73-containing tRNAPyl will enable future efforts to engineer PylRS systems for further genetic code expansion.
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    Directed Evolution of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase Generates a Hyperactive and Highly Selective Variant
    (Frontiers Media, 2022-03-09) Fischer, Jonathan T.; Söll, Dieter; Tharp, Jeffery M.; Biochemistry and Molecular Biology, School of Medicine
    Pyrrolysyl-tRNA synthetase (PylRS) is frequently used for site-specific incorporation of noncanonical amino acids (ncAAs) into proteins. Recently, the active site of Methanomethylophilus alvus PylRS (MaPylRS) has been rationally engineered to expand its substrate compatibility, enabling the incorporation of difficult ncAAs. However, mutations beyond the active site that enhance the enzymatic properties of MaPylRS have not been reported. We utilized phage-assisted non-continuous evolution (PANCE) to evolve MaPylRS to efficiently incorporate N ε-Boc-l-lysine (BocK). Directed evolution yielded several mutations outside of the active site that greatly improve the activity of the enzyme. We combined the most effective mutations to generate a new PylRS variant (PylRSopt) that is highly active and selective towards several lysine and phenylalanine derivatives. The mutations in PylRSopt can be used to enhance previously engineered PylRS constructs such as MaPylRSN166S, and PylRSopt is compatible in applications requiring dual ncAA incorporation and substantially improves the yield of these target proteins.
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    Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons
    (American Chemical Society, 2021) Tharp, Jeffery M.; Vargas-Rodriguez, Oscar; Schepartz, Alanna; Söll, Dieter; Biochemistry and Molecular Biology, School of Medicine
    We recently described an orthogonal initiator tRNA (itRNATy2) that can initiate protein synthesis with noncanonical amino acids (ncAAs) in response to the UAG nonsense codon. Here, we report that a mutant of itRNATy2 (itRNATy2AUA) can efficiently initiate translation in response to the UAU tyrosine codon, giving rise to proteins with an ncAA at their N-terminus. We show that, in cells expressing itRNATy2AUA, UAU can function as a dual-use codon that selectively encodes ncAAs at the initiating position and predominantly tyrosine at elongating positions. Using itRNATy2AUA, in conjunction with its cognate tyrosyl-tRNA synthetase and two mutually orthogonal pyrrolysyl-tRNA synthetases, we demonstrate that UAU can be reassigned along with UAG or UAA to encode two distinct ncAAs in the same protein. Furthermore, by engineering the substrate specificity of one of the pyrrolysyl-tRNA synthetases, we developed a triply orthogonal system that enables simultaneous reassignment of UAU, UAG, and UAA to produce proteins containing three distinct ncAAs at precisely defined sites. To showcase the utility of this system, we produced proteins containing two or three ncAAs, with unique bioorthogonal functional groups, and demonstrate that these proteins can be separately modified with multiple fluorescent probes.
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    Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression
    (National Academy of Science, 2023) Jiang, Han-Kai; Ambrose, Nicole L.; Chung, Christina Z.; Wang, Yane-Shih; Söll, Dieter; Tharp, Jeffery M.; Biochemistry and Molecular Biology, School of Medicine
    Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein-protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.