Fig. 1. Bioorthogonal labeling of TARPs.

a Schematic overview of click chemistry labeling via genetic code expansion. Amber mutants of ncAA-tagged γ2 and γ8 were designed by standard site-direct mutagenesis to incorporate the ncAAs carrying a TCO*A for click labeling. Protein expression occurs through endogenous and orthogonal tRNA-synthetases in the presence of appropriate tRNAs. Labeling of ncAA-tagged proteins occurs through a catalysis-free reaction between TCO*A and tetrazine-functionalized dyes via the strain-promoted inverse electron-demanding Diels-Alder cycloaddition (SPIEDAC) reaction. (b) Sequence alignment of the first extracellular loops of γ2 and γ8 from rattus norvegicus. Amber substitution mutations are represented in red. c, d Representative confocal images of living HEK293T cells co-expressing PylRS/4xtRNA^Pyl and c γ2 S44*::eGFP or d γ8 S72*::eGFP in the absence (upper) or presence of 250 μM TCO*A (lower) stained with 1.5 μM Pyr-Tet-ATTO643. Scale bar: 20 μm. Images are representative of three independent experiments. e, f Representative spinning disk confocal images of living dissociated hippocampal neurons co-expressing eGFP, Tet3G/tRNA^Pyl and e pTRE3G-BI PylRS/γ2 S44* or f pTRE3G-BI PylRS/γ8 S72* in the presence of 250 μM TCO*A and 100 ng mL⁻¹ doxycycline labeled with 0.5 μM Pyr-Tet-ATTO643. Bottom panels, magnified views of segments of dendrites highlighted in the eGFP panel (dashed yellow boxes) of the overview images showing the distribution of γ2 S44* and γ8 S72*. Scale bar: 20 μm (overview images) and 5 μm (magnified images). All example images are representative of at least three independent preparations.