Tyrosine nitration has served as a major biomarker for oxidative stress and is present in high abundance in over 50 disease pathologies in humans. While data mounts on specific disease pathways from specific sites of tyrosine nitration, the role of these modifications is still largely unclear. Strategies for installing site-specific tyrosine nitration in target proteins in eukaryotic cells, through routes not dependent on oxidative stress, would provide a powerful method to address the consequences of tyrosine nitration. Developed here is a Methanosarcina barkeri aminoacyl-tRNA synthetase/tRNA pair that efficiently incorporates nitrotyrosine site-specifically into proteins in mammalian cells. We demonstrate the utility of this approach to produce nitrated proteins identified in disease conditions by producing site-specific nitroTyr-containing manganese superoxide dismutase and 14-3-3 proteins in eukaryotic cells.

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Assembling nanobodies (Nbs) into polyvalent multimers is a powerful strategy for improving the effectiveness of Nb-based therapeutics and biotechnological tools. However, generally effective approaches to Nb assembly are currently restricted to the amino or carboxyl termini, greatly limiting the diversity of Nb multimer topologies that can be produced. Here, we show that reactive tetrazine groups—site-specifically inserted by genetic code expansion at Nb surface sites—are compatible with Nb folding and function, enabling Nb assembly at any desired point. Using two anti–SARS-CoV-2 Nbs with viral neutralization ability, we created Nb homo- and heterodimers with improved properties compared with conventionally linked Nb homodimers, which, in the case of our tetrazine-conjugated trimer, translated into enhanced viral neutralization. Thus, this tetrazine-based approach is a generally applicable strategy that greatly increases the accessible range of Nb assembly topologies, and thereby adds the optimization of topology as an effective avenue to generate Nb assemblies with improved efficacy.

A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.

E dward Lemke and his team have recently reported the