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Genetic code expansion technology allows for the use of noncanonical amino acids (ncAAs) to create semisynthetic organisms for both biochemical and biomedical applications. However, exogenous feeding of chemically synthesized ncAAs at high concentrations is required to compensate for the inefficient cellular uptake and incorporation of these components into proteins, especially in the case of eukaryotic cells and multicellular organisms. To generate organisms capable of autonomously biosynthesizing an ncAA and incorporating it into proteins, we have engineered a metabolic pathway for the synthesis of O-methyltyrosine (OMeY). Specifically, we endowed organisms with a marformycins biosynthetic pathway-derived methyltransferase that efficiently converts tyrosine to OMeY in the presence of the co-factor S-adenosylmethionine. The resulting cells can produce and site-specifically incorporate OMeY into proteins at much higher levels than cells exogenously fed OMeY. To understand the structural basis for the substrate selectivity of the transferase, we solved the X-ray crystal structures of the ligand-free and tyrosine-bound enzymes. Most importantly, we have extended this OMeY biosynthetic system to both mammalian cells and the zebrafish model to enhance the utility of genetic code expansion. The creation of autonomous eukaryotes using a 21st amino acid will make genetic code expansion technology more applicable to multicellular organisms, providing valuable vertebrate models for biological and biomedical research.

Photoactivatable fluorophores are emerging optical probes for biological applications. Most photoactivatable fluorophores are relatively large in size and need to be activated by ultraviolet light; this dramatically limits their applications. To introduce photoactivatable fluorophores into proteins, recent investigations have explored several protein-labeling technologies, including fluorescein arsenical hairpin (FlAsH) Tag, HaloTag labeling, SNAPTag labeling, and other bioorthogonal chemistry-based methods. However, these technologies require a multistep labeling process. Here, by using genetic code expansion and a single sulfur-for-oxygen atom replacement within an existing fluorescent amino acid, we have site-specifically incorporated the photoactivatable fluorescent amino acid thioacridonylalanine (SAcd) into proteins in a single step. Moreover, upon exposure to visible light, SAcd can be efficiently desulfurized to its oxo derivatives, thus restoring the strong fluorescence of labeled proteins.

While most organisms utilize 20 canonical amino acid building blocks for protein synthesis, adding additional candidates to the amino acid repertoire can greatly facilitate the investigation and manipulation of protein structures and functions. In this study, we report the generation of completely autonomous organisms with a 21st ncAA, 5-hydroxytryptophan (5HTP). Like 20 canonical amino acids, 5-hydroxytryptophan can be biosynthesized in vivo from simple carbon sources and is subsequently incorporated into proteins in response to the amber stop codon. Using this unnatural organism, we have prepared a single-chain immunoglobulin variable fragment conjugated with a fluorophore and demonstrated the utility of these autonomous cells to monitor oxidative stress. Creation of this and other cells containing the 21st amino acid will provide an opportunity to generate proteins and organisms with novel activities, as well as to determine the evolutionary consequences of using additional amino acid buildings.

Site-specific antibody conjugates with a well-defined structure and superb therapeutic index are of great interest for basic research, disease diagnostics, and therapy. Here, we develop a novel proximity-induced antibody conjugation strategy enabling site-specific covalent bond formation between functional moieties and native antibodies without antibody engineering or additional UV/chemical treatment. A high conjugation efficiency and specificity was achieved with IgGs from different species and subclasses. The utility of this approach was demonstrated by site-specific conjugation of the small-molecule fluorophore to a native antibody and in vitro characterization of its activities.

Precision modification of antibodies with bone-targeting moieties unleashes their potential for the treatment of bone metastases., Antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone microenvironment has proved to be difficult due to the relatively low vascularization of bone tissue and the presence of physical barriers. Here, we have used an innovative bone-targeting (BonTarg) technology to generate a first-in-class bone-targeting antibody. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to therapeutic antibodies. Bisphosphonate modification of these antibodies results in the delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In xenograft mice models, this strategy provides enhanced inhibition of bone metastases and multiorgan secondary metastases that arise from bone lesions. Specific delivery of therapeutic antibodies to the bone, therefore, represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases.

Site-specific introduction of bioorthogonal handles into biomolecules provides powerful tools for studying and manipulating the structures and functions of proteins. Recent advances in bioorthogonal chemistry demonstrate that tetrazine-based bioorthogonal cycloaddition is a particularly useful methodology due to its high reactivity, biological selectivity, and turn-on property for fluorescence imaging. Despite its broad applications in protein labeling and imaging, utilization of tetrazine-based bioorthogonal cycloaddition has been limited to date by the requirement of a hydrophobic strained alkene reactive moiety. Circumventing this structural requirement, we report the site-specific incorporation of noncanonical amino acids (ncAAs) with a small isocyanide (or isonitrile) group into proteins in both bacterial and mammalian cells. We showed that under physiological conditions and in the absence of a catalyst these isocyanide-containing ncAAs could react selectively with tetrazine molecules via [4 + 1]-cycloaddition, thus providing a versatile bioorthogonal handle for site-specific protein labeling and protein decaging. Significantly, these bioorthogonal reactions between isocyanides and tetrazines also provide a unique mechanism for the activation of tetrazine-quenched fluorophores. The addition of these isocyanide-containing ncAAs to the list of 20 commonly used, naturally occurring amino acids expands our repertoire of reagents for bioorthogonal chemistry, therefore enabling new biological applications ranging from protein labeling and imaging studies to the chemical activation of proteins.

Genetically site-specific introduction of noncanonical amino acids (ncAAs) for protein conjugation generally requires incorporation through exogenous feeding of chemically synthesized ncAAs. We developed a p-amino-phenylalanine (pAF)-based relay system that enables site-specific functionalization of proteins without chemical synthesis of the building blocks. pAF was biosynthesized under optimized conditions, followed by site-specific incorporation into a specific protein residue. The resulting protein was ready for functionalization using an oxidative conjugation reaction. We demonstrated the use of this relay system by preparing a fluorophore-labeled anti-HER2 single-chain variable fragment antibody for fluorescent imaging.