Tuning the reactivity of substrate for SNAP-tag expands its application for recognition-driven DNA-protein conjugation.

Recognition-driven modification has been emerging as a novel approach to modify biomolecular targets of interest site-specifically and efficiently. To this end, the protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA, which constitute both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling the DNA recognition by DBD and the chemoselective reaction of protein tag could provide a highly efficient sequence-specific reaction. However, combination of MA consisting of a reactive protein-tag and its substrate, e.g., SNAP-tag and benzyl guanine (BG), revealed rather non-selective reaction with DNA. Here, we designed new substrates of SNAP-tag to realize the sequence-selective rapid closslinking reaction by MAs with SNAP-tag. The reactions of substrates to SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables distinctive orthogonality of SNAP-tag against CLIP-tag to achieve the orthogonal DNA-protein cross-linking reaction by six unique MAs.