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. Author manuscript; available in PMC: 2020 Dec 3.
Published in final edited form as: J Mol Biol. 2020 Apr 8;432(10):3127–3136. doi: 10.1016/j.jmb.2020.03.033

Figure 3.

Figure 3.

Use of cpRAPID to design Chessbody for ligand-gated antigen recognition in living cells. (a) Design of a Chessbody that recognizes mCherry upon addition of rapamycin. The N- and C-terminal fragments of a split nanobody (LaM8; split between residues S64 and V65) was fused to cpFRB and FKBP, respectively. The chemical induced dimerization of the cpRAPID system facilitates the reassembly of a functional Chessbody, which restores the antibody–antigen binary interaction between mitochondria (mito)-anchored mCherry (antigen) and the LaM8-specific Chessbody composed of GFP-cpFRB-LaM8(N) and FKBP-LaM8(C). (b) Representative confocal images of HeLa cells co-expressing the indicated Chessbody components (green) and the antigen (red; AKAP1-mCherry or Mito-mCh) before and after rapamycin (600 nM) treatment for 30 min. The scale bar represents 5 μm. (c) Design of a GFP-specific Chessbody made of mCh-cpFRB-LaGFP(N) and FKBP-LaGFP(C), which exhibits rapamycin-inducible binding toward the mitochondria-tethered antigen (NTOM20-GFP). (d) Representative confocal images of HeLa cells co-expressing a GFP-recognizing Chessbody (red; mCh-cpFRB-LaGFP(N) + FKBP-LaGFP(C)) and mitochondria-tethered GFP (green; NTOM20-GFP or Mito-GFP) before and after rapamycin treatment as did in (b). The scale bar represents 5 μm. (e) Real-time monitoring of rapamycin (600 nM) induced restoration of Chessbody–antigen interaction using the indicated FKBP–FRB/cpFRB pairs. The Pearson’s correlation between GFP and mCherry signals at mitochondria was used to quantitate the degrees of colocalization. The corresponding half-lives (t1/2) using a single-phase decay fitting were as follows: 20.0 ± 8.2 min (FRB), 18.0 ± 4.0 min (cpFRB1), and 7.8 ± 1.8 min (cpFRB2). For cpFRB2, if using a two-phase decay fitting, the half-lives were as follows: t1/2, fast = 1.5 ± 0.1 min and t1/2, slow = 14.1 ± 1.5 min. n = 10–23 cells from three independent assays (mean ± s.e.m.). (f) Quantification of Pearson's correlation for the indicated Chessbodies before and after rapamycin treatment (as shown in (b) and (d)). n = 10–23 cells from three independent assays (mean ± s.e.m.).