LOV2 insertion sites mapped to the 3D structure of an anti-SH2Abi monobody (PDB entry: 3T04). Yellow circles represent the three CDR (complementarity-determining region)-like loop regions that mediate moonbody-target recognition.ī. Light-dependent shuttling of moonbody between NE and the nucleoplasm (quantified as the NE/NP ratio of mCh signals) is monitored. A photoswitch LOV2 is inserted into selected loop regions to enable photo-inducible target recognition in a reversible manner. Schematic depicting the design of light-switchable monobody (designated “moonbody”) and the nuclear envelope (NE) translocation assay used for screening. Design and optimization of light-switchable monobody (moonbody).Ī. Contrariwise, insertion at flexible loop regions opposing the CDR-like areas, such as S1, S3, and S5, caused varying degrees of light-induced dissociation of moonbody from NE, with the S5 construct showing the highest dynamic change and hence used for further optimization ( Figure 1d, and Supplementary Figure 1a-c).įigure 1. The insertion of LOV2 at Site 2 (BC loop) led to an appreciable increase (<10%) of nucleoplasmic moonbody toward NE-SH2 ( Figure 1d, and Supplementary Figure 1b-c). Insertion of LOV2 at Site 4 (DE loop) abolished the moonbody-target interaction regardless of the presence of light ( Supplementary Figure 1b-c), likely owing to the disruption of the antigen-binding pocket. To visualize the light-dependent changes in moonbody-SH2 association in live cells, we anchored the SH2 domain to the nuclear envelope (NE) via fusion with mEmerald-lamin A, and co-expressed the engineered moonbody as a cytosolic protein with partial distribution in the nucleoplasm (NP). We envisioned that light-induced conformational changes in LOV2 would allosterically modulate the moonbody-target interaction, thereby permitting photoswitchable control of the moonbody. Six insertion sites at exposed loop regions ( Supplementary Figure 1a) were selected, with three situated in the antigen-recognizing BC, DE, FG loops (the equivalent of complementarity-determining regions (CDRs) seen in an antibody) and the other three at the opposing loops ( Figure 1a-c). We first set out to design a light-controllable monobody (termed moonbody) by inserting the LOV2 photoswitch into a fibronectin type III domain (FN3) scaffold that specifically recognizes the Src Homology 2 (SH2) domain of Abelson tyrosine kinase (Abl). With these smart sunbodies or moonbodies, we demonstrate their wide applications in the remote control of protein localization, cell death, transcriptional programming and precise base editing.Ģ.1 Design of Moonbody as Light-Controllable Antibody Mimetics ![]() ![]() By optimizing LOV2 insertion sites and paralleled insertion of two LOV2 modules into a single sunbody, we significantly enhanced the dynamic range of light-inducible response when compared to the existing OptoNB. We greatly improved the perfomance of our light-controlled moonbodies by optimzing the LOV2 insertion in the EF loop rather than the DE loop as described in the OptoMB tool. The light-oxygen-voltage domain 2 (LOV2) derived from Avena Sativa phototropin 1 has been engineered into intrabodies to confer allosteric control of protein activity by light. Nontheless, these engineered intrabodies exhibit a relatively slow activation kinetics or suffer from a relatively low or modest dynamic range of light-induced changes.īearing these unmet needs in mind, we introduce herein a set of smart monobodies and nanobodies that respond to light within seconds, in the form of ON-switches (sunbody) or OFF-switches (moonbody). Recent engineering efforts have led to the generation of several classes of chemically or light-dependent single-domain antibodies, either based on split nanobodies (optobody) or hybrid proteins that utilize a photosensitive switch (OptoNB and OptoMB) or circularly permuted bacterial dihydrofolate reductase (LAMA). Single-domain intrabodies and their mimetics such as monobodies or nanobodies rival conventional antibodies by their substantially smaller sizes (12-15 kDa vs 150-160 kDa), freedom from disulfide-bond formation, and ease of in vitro and in cellular expression. Intracellular single-domain antibodies (intrabodies) and their mimetics derived from synthetic protein scaffolds, as most notably exemplified by nanobodies and monobodies, are gaining wide use in cell biology, structural biology, synthetic immunology, and theranostics.
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