Extended Data Fig. 3: The mechanism of action of SMART-SpyCatcher.

a, K562^HER2+/EGFR+ cells were treated with αHER2-SpyN (100 nM) and SpyC-αEGFR (100 nM) for 2 hr, followed by SpyTag003 labeled with Alexa Fluor 594 (SpyTag003-AF594, 100 nM) for 20 min. SMART-SpyCatcher employed eNrdJ-1^cage. Following washing, the live cells were analyzed by confocal microscopy. Cell nuclei were stained with Hoechst, while HER2 and EGFR were tagged with eGFP and iRFP, respectively. The cells in row 1 were treated as described above, whereas reaction conditions in the subsequent experiments were supplemented with DARPins targeting HER2 and EGFR (500 nM each, row 2), used an inactivated version of NrdJ-1N^cage (Cys1-alkylated, row 3), or were supplemented with unlabeled SpyTag003 (500 nM, row 4). Scale bar equals 20 μm. All subsequent panels apply similar reaction and analysis conditions as in panel a (row 1) using the SMART-SpyCatcher (eNrdJ-1^cage) system with any alterations as noted. b, Western blot analysis of K562^HER2+/EGFR+ cells treated with αHER2-SpyN and SpyC-αEGFR (identified by FLAG and Myc respectively), and SpyTag003 labeled with biotin (SpyTag003-biotin; identified by Strep-800). The inactivated version of NrdJ-1N^cage (Cys1-alkylated) was used as a negative control. c, Experiments were performed on K562^HER2+/EGFR+ cells with the SMART-SpyCatcher (eNrdJ-1^cage) [HER2 AND EGFR] system, and either SpyTag003-AF594 or inactive SpyTag003^D117A labeled with Alexa Fluor 594 (SpyTag003^D117A-AF594). Scale bar equals 20 μm. d, K562^HER2+/EGFR+ cells were treated with SMART-SpyCatcher (100 nM, eNrdJ-1^cage) in buffer (DPBS, 1% w/v BSA, 2 mM CaCl₂), RPMI 1640 medium (cystine-free), or in medium supplemented with 5% v/v fetal bovine serum (5% v/v FBS) for 2 hr, followed by SpyTag003-AF594 for 20 min. Further image analysis was performed as described above. Scale bar equals 20 μm. e, Flow cytometry analysis of a mixed population consisting of equal amounts of K562 (wildtype), K562^HER2+, K562^EGFR+, and K562^HER2+/EGFR+ cells following treatment with SMART-SpyCatcher (eNrdJ-1^cage) operating through the AND logic indicated. The added SpyN and SpyC pairs (i.e., the targeting DARPins employed in the constructs) are indicated at the bottom. Data are presented as the mean of the AF594 median fluorescence intensities (MFI) from flow cytometry analysis with error bars signifying the standard error mean (n = 3 independent biological replicates; see Supplementary Table 13 for statistical one-way ANOVA followed by Dunnett’s test). f, The mixed K562 population described above were treated with αHER2-SpyN (100 nM) and SpyC-αEGFR (100 nM) employing eNrdJ-1^cage or the uncaged split intein NrdJ-1, for 2 hr, followed by SpyTag003-AF594 (100 nM) for 20 min. Further image analysis was performed as described above. Scale bars are equal to 20 μm. Data shown in panels a-d, and f are representative of two independent experiments.