Extended Data Fig. 1: Efficient decoration of an N₃ tag on the inner plasma membrane (IPM).

a, Chemical structures of m^Dc-N₃ and IPM-N₃. b, The membrane penetration kinetics of Silorhodamine (SiR)-DBCO. Left: Representative confocal images of HeLa cells incubated with 10 μM SiR-DBCO at 37 °C for different time spans (scale bar, 10 µm). Right: Summary of time-dependent SiR fluorescence intensity (n = 30, 30, and 31 cells for the left to right groups, respectively). c, Schematic illustration of the experimental workflow for construction of IPM-N₃. d, Representative confocal images of SiR-DBCO-pretreated HeLa cells processed with or without 10 μM m^Dc-N₃ at RT for 10 min, and then incubated in fresh culture medium for different time spans (scale bar, 10 µm). e, Schematic illustration of the experimental workflow for quantification of SiR-DBCO within cells. f, Left: Construction of the standard calibration curve of SiR-DBCO. Right: Fluorescence spectra of the lysate of HeLa cells after treatment with 10 μM SiR-DBCO at 37 °C for 60 min. g, Quantification of IPM-DNs. Construction of the standard calibration curve of DN_Cy5 in buffer solution (Standard curve 1), DN_Cy5 on the outer membrane of cells (Standard curve 2), and grey value of Cy5 in confocal microscope images (Standard curve 3). h, Summary of time-dependent changes of SiR fluorescence ratio between the plasma membrane and whole cell in SiR-DBCO-pretreated HeLa cells incubated with m^Dc-N₃ (Left to right groups: n = 10, 10, 13, and 12 cells for m^Dc-N₃ treated group; n = 12, 12, 13, and 13 cells for control group without m^Dc-N₃ treatment). i, Representative confocal images of HeLa cells treated with m^Dc-N₃ for 45 min (scale bar, 10 μm, green fluorescence = DIO for plasma membrane staining). j, Cellular distribution of the N₃ tag. Representative confocal images of HeLa cells processed with SiR-DBCO/m^Dc-N₃ and then stained with 100 nM Golgi Apparatus (GA) tracker, Endoplasmic Reticulum (ER) tracker, or Mitochondria (Mito) tracker (scale bar, 10 µm). k, Golgi apparatus-mediated metabolic process of m^Dc-N₃. Representative confocal images of HeLa cells pretreated with 10 μM palmitoylation inhibitor 2-bromopalmitate (2-BP) (upper) or 20 μM Golgi disrupter Brefeldin A (BFA) (lower) at 37 °C for 60 min, and then processed with SiR-DBCO/m^Dc-N₃ (scale bar, 10 µm). l, The IPM orientation of the N₃ tag. Representative confocal images of mSA-EGFP-transfected HeLa cells processed with m^Dc-N₃, and then incubated with DBCO-PEG₄-Biotin at RT for 30 min (scale bar, 10 μm (upper) and 2.5 μm (lower)). Statistical data are presented as mean ± s.d. (b) or mean ± s.e.m. (h), with n as randomly selected cells from 3 biological repeats. For panel d and i-l, 3 independent experiments are repeated with similar results.

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