Figure 4.

Using TAMM condensation, CuAAC, and tetrazine ligation for multicolor fluorescent labeling. (A) Structure of unnatural amino acids CysK, PPA, BCNK, and HexC. (B) Substrate scope of the orthogonal aminoacyl-tRNA synthetases. (C) Structure of FITC-N₃ and Pyranine-tetrazine. (D) Using three unnatural amino acids to control the bioorthogonal reactions for labeling three target proteins on different HEK293T cells. (E) Using two sequence specific proteases for iterative TAMM condensation in combination with tetrazine ligation and CuAAC for labeling four different target proteins on HEK293T cells. (D,E) Schematic presentation of the experimental flow is shown at the top. Representative confocal microscopy images are shown at the bottom. Two other sets of data are shown in Figures S32 and S33. Scale bar = 20 μm.