Fig. 2: Receptor activation via a bioorthogonal Tz-TCO ligation reaction.

a Chemical structures of Tetrazine-5-Fluorescein (Tz-5-fluorescein) and the trans-cyclooctene (TCO) reactive handle depicted as conjugated to BSA (TCO-BSA). b Schematic depiction of the bio-orthogonal ligation-mediated immobilization of Tz-5-fluorescein upon its reaction to surface-adsorbed TCO-BSA. Generation of the immobilized fluorescein ligand results in productive αFITC(E2)-SynNotch activation and H2B-mCherry reporter expression. c Dose-dependent analysis of Tz-5-fluorescein-mediated H2B-mCherry expression from clonal αFITC(E2)-SynNotch cells grown in TCO-BSA coated microwells. Traces represent normalized densities of three independent treatments at varying doses are shown. The dashed black line indicates the H2B-mCherry+ threshold set based on analysis of control HEK293-FT cells; solid black lines indicate median mCherry intensities for each condition. d Median mCherry emission intensities from untreated and Tz-5-fluorescein-treated cells grown with or without immobilized TCO-BSA from (c). Three independent drug treatments (n = 3 >5000 cells analyzed per replicate); Data presented as mean values +/- standard deviation and analyzed with two-way ANOVA (ligand and drug concentration). Labeled NS, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data and P values provided in Source Data file. e Fluorescence images of clonal HEK293-FT (UAS:H2B-mCherry) reporter cells expressing αFITC(E2)-SynNotch-mTurq2, as treated under the indicated conditions with 2 nM Tz-5-Fluorescein. Scale bar = 100 µm. f Immunoblot detection of signaling-induced H2B-mCherry levels. Tz-fluorescein concentrations were tested between 2 pM and 200 nM, with variation between adjacent lanes by a factor of 10-fold. For both (e) and (f), treatment with gamma secretase inhibitor (GSI, DAPT at 10 μM) led to diminished Tz-5-fluorescein-induced reporter H2B-mCherry levels. Experiments in (e) and (f) were repeated independently with similar results. The cartoon cells in (b) were created in BioRender⁸⁷.