Figure 1.

Design and optimization of light-switchable monobody (moonbody). a) Schematic depicting the design of light-switchable monobody (designated “moonbody”) and the nuclear envelope (NE) translocation assay used for screening. A photoswitch LOV2 is inserted into selected loop regions to enable photo-inducible target recognition in a reversible manner. Light-dependent shuttling of moonbody between NE and the nucleoplasm (quantified as the NE/NP ratio of mCh signals) is monitored. Yellow circles represent the three CDR (complementarity-determining region)-like loop regions that mediate moonbody–target recognition. b) LOV2 insertion sites mapped to the 3D structure of an anti-SH2_Abl monobody (PDB entry: 3T04). Even-numbered insertion sites were created in the target-recognition loops, whereas odd-numbered sites were located opposite to the antigen-recognizing BC/DE/FG loops. c) 2D topology representation of an anti-SH2 monobody, with the insertion sites indicated by circles. The monobody-LOV2 junction regions for S5 or its variants were shown below the cartoon. See Figure S1, Supporting Information, for detailed sequence information of all constructs tested in the study. d) Quantification of light-dependent responses (as the NE/NP ratio) of moonbody variants. See Figure S1, Supporting Information, for representative images. Insertion at Site 5 (S5) led to the highest light-induced change. n = 6–25 cells. Data are shown as mean ± SEM. e) Representative confocal images of a HeLa cell coexpressing an anti-SH2 moonbody (mCh-tagged variant S5.1; red) and NE-tethered SH2 domain of Abl kinase (NE tethered-antigen or abbreviated as NE-Ag; green) in the dark or after light illumination for 10 s. Scale bar = 10 μm.