Exploration and Application of DNA-Binding Proteins to Make a Versatile DNA–Protein Covalent-Linking Patch (D-Pclip): The Case of a Biosensing Element

DNA–protein complexes are attractive components with broad applications in various research fields, such as DNA aptamer–enzyme complexes as biosensing elements. However, noncovalent DNA–protein complexes often decrease detection sensitivity because they are highly susceptible to environmental conditions. In this study, we developed a versatile DNA–protein covalent-linking patch (D-Pclip) for fabricating covalent and stoichiometric DNA–protein complexes. We comprehensively explored the database to determine the DNA-binding ability of the candidates and selected UdgX as the only uracil-DNA glycosylase known to form covalent bonds with DNA via uracil, with a binding efficiency >90%. We integrated a SpyTag/SpyCatcher protein-coupling system into UdgX to create a universal and convenient D-Pclip. The usability of D-Pclip was shown by preparing a stoichiometric model complex of a hemoglobin (Hb)-binding aptamer and glucose oxidase (GOx) by mixing at 4 °C. The prepared aptamer-GOx complexes detected Hb in a dose-dependent manner within the clinically required detection range in buffer and human serum without any washing procedures. D-Pclip covalently connects any uracil-inserted DNA sequence and any SpyCatcher-fused protein stoichiometrically; therefore, it has a high potential for various applications.