Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation |
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Authors: | Zhengxiao Zhang Prof. Eiji Nakata Dr. Huyen Dinh Dr. Masayuki Saimura Prof. Arivazhagan Rajendran Prof. Kazunari Matsuda Prof. Takashi Morii |
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Affiliation: | Institute of Advanced Energy, Kyoto University Uji, Kyoto, 6110011 Japan |
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Abstract: | Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs. |
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Keywords: | DNA sequence specificity conjugation orthogonality protein tags selectivity |
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