Mutually Orthogonal Nonsense-Suppression Systems and Conjugation Chemistries for Precise Protein Labeling at up to Three Distinct Sites

2019/03/25 by James S. Italia, Partha Sarathi Addy, Sarah B. Erickson +3 · 110 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · Medicine · #Amino acid #Aminoacyl tRNA synthetase #Biochemistry #Bioconjugation #Biology #Bioorthogonal chemistry #Chemical Synthesis and Analysis #Chemical biology #Chemistry #Click Chemistry and Applications #Click chemistry #Combinatorial chemistry #Computational biology #Enzyme #Monoclonal and Polyclonal Antibodies Research #Protein engineering #RNA #Transfer RNA

paper · open access · doi:10.1021/jacs.8b12954

published in Journal of the American Chemical Society 141(15), 6204-6212 (American Chemical Society)

openalex publication_date 2019/03/25 · openalex created_date 2025/10/10 · openalex updated_date 2026/08/01

Abstract

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into a protein is an emerging technology with tremendous potential. It relies on mutually orthogonal engineered aminoacyl-tRNA synthetase/tRNA pairs that suppress different nonsense/frameshift codons. So far, up to two distinct ncAAs have been incorporated into proteins expressed in E. coli, using archaea-derived tyrosyl and pyrrolysyl pairs. Here we report that the E. coli derived tryptophanyl pair can be combined with the archaeal tyrosyl or the pyrrolysyl pair in ATMW1 E. coli to incorporate two different ncAAs into one protein with high fidelity and efficiency. By combining all three orthogonal pairs, we further demonstrate simultaneous site-specific incorporation of three different ncAAs into one protein. To use this technology for chemoselectively labeling proteins with multiple distinct entities at predefined sites, we also sought to identify different bioconjugation handles that can be coincorporated into proteins as ncAA-side chains and subsequently functionalized through mutually compatible labeling chemistries. To this end, we show that the recently developed chemoselective rapid azo-coupling reaction (CRACR) directed to 5-hydroxytryptophan (5HTP) is compatible with strain-promoted azide-alkyne cycloaddition (SPAAC) targeted to p-azidophenylalanine (pAzF) and strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) targeted to cyclopropene-lysine (CpK) for rapid, catalyst-free protein labeling at multiple sites. Combining these mutually orthogonal nonsense suppression systems and the mutually compatible bioconjugation handles they incorporate, we demonstrate site-specific labeling of recombinantly expressed proteins at up to three distinct sites.

Citations

• A Strain-Promoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems
• Genetic Encoding of Bicyclononynes and trans-Cyclooctenes for Site-Specific Protein Labeling in Vitro and in Live Mammalian Cells via Rapid Fluorogenic Diels–Alder Reactions
• Tetrazine Ligation: Fast Bioconjugation Based on Inverse-Electron-Demand Diels−Alder Reactivity

Cited by

• Copper-catalysed azide–alkyne cycloaddition on live M13 bacteriophage for expanding the molecular diversity of phage-displayed peptide libraries
• Aminoacyl-tRNA Synthetases and tRNAs for an Expanded Genetic Code: What Makes them Orthogonal? [europepmc]
• Isomeric triazines exhibit unique profiles of bioorthogonal reactivity. [europepmc]
• New codons for efficient production of unnatural proteins in a semisynthetic organism. [europepmc]
• Engineered triply orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids. [europepmc]
• Synthetic Biological Circuits within an Orthogonal Central Dogma. [europepmc]
• Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides. [europepmc]
• Using Genetic Code Expansion for Protein Biochemical Studies. [europepmc]
• Chemical modifications of proteins and their applications in metalloenzyme studies. [europepmc]
• Genetic Encoding of Three Distinct Noncanonical Amino Acids Using Reprogrammed Initiator and Nonsense Codons. [europepmc]
• Sense codon reassignment enables viral resistance and encoded polymer synthesis. [europepmc]
• Bioorthogonal Reactions of Triarylphosphines and Related Analogues. [europepmc]
• Developing bioorthogonal probes to span a spectrum of reactivities. [europepmc]
• A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. [europepmc]
• Precision Modification of Native Antibodies. [europepmc]
• Multiplex suppression of four quadruplet codons via tRNA directed evolution. [europepmc]
• Biosynthesis and Genetic Incorporation of 3,4-Dihydroxy-L-Phenylalanine into Proteins in Escherichia coli. [europepmc]
• Noncanonical amino acids as doubly bio-orthogonal handles for one-pot preparation of protein multiconjugates. [europepmc]
• An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells. [europepmc]
• Site-specific dual encoding and labeling of proteins via genetic code expansion. [europepmc]
• Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo. [europepmc]
• Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNA Pyl pairs. [europepmc]
• Adding New Chemistries to the Central Dogma of Molecular Biology. [europepmc]
• Noncanonical Amino Acids in Biocatalysis. [europepmc]
• Cracking the Code: Reprogramming the Genetic Script in Prokaryotes and Eukaryotes to Harness the Power of Noncanonical Amino Acids. [europepmc]
• Engineering Pyrrolysine Systems for Genetic Code Expansion and Reprogramming. [europepmc]

Related

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• Engineered orthogonal translation systems from metagenomic libraries expand the genetic code
• Expanding and Reprogramming the Genetic Code of Cells and Animals
• Hijacking a bacterial ABC transporter for genetic code expansion