Quantitative Protein Labeling in Live Cells by Controlling the Redox State of Encoded Tetrazines

The site-specific attachment of fluorophores, probes, or drugs to proteins in living systems is critical for advancing our understanding of biology and drug development. The site-specific encoding of 1,2,4,5-tetrazine (Tet) residues into target proteins provides the rapid kinetics and stability required for quantitative labeling in living cells, a property that is increasingly desirable as the resolution and specificity of imaging increases. Here, we adapt a common gel-shift assay to create a "PEG Chaser assay" for evaluating labeling completeness in living cells by "chasing" in-cell Tet reactions with an in vitro reaction with a TCO-PEG₅₀₀₀ polymer; then, a gel shift distinguishes proteins that did not react in cells from those that did. We apply this to observe that encoded Tets exist in an equilibrium between oxidized (Tz) and reduced (DHTz) forms in living cells. We further show how a recently developed photooxidation treatment can convert the nonreactive DHTz Tet-protein to the reactive Tz form and enables its rapid, quantitative in-cell labeling. We then develop genetic code expansion machinery for encoding two new Tet ncAAs with different redox potentials and show how the tuning of the Tet redox is a useful variable for controlling the reactivity of Tet ncAAs. Specifically, the new Tet3H ncAA enables photoactivatable labeling and complete protein labeling in living cells in 5 min. This in-depth evaluation of the impact of the intracellular reducing environment on Tet reactivity and the demonstration of controlling Tet redox in living cells expands the utility of encodable Tet in living cells.