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NanoBondy principle. (A) NeissLock chemistry. Upon the addition of calcium, the self-processing module (SPM) activates autoproteolysis at the aspartate–proline bond. This step generates a highly reactive aspartyl anhydride, which undergoes nucleophilic attack by a nearby nucleophilic amino acid or water. Fusing SPM to a binder (purple) thereby allows inducible covalent coupling to a target protein (green). (B) NanoBondy design. A nanobody (purple) employs complementarity-determining regions (CDRs) close to the N-terminus to bind its target (green). A regular nanobody can be engineered into a covalently reacting NanoBondy by inclusion of a flexible linker and disulfide clamp (magenta) to hold the reactive D (cyan) of SPM (orange) near the target, allowing anhydride-mediated covalent conjugation to the target after calcium activation.