Exploration and Application of DNA-Binding Proteins to Make a Versatile DNA-Protein Covalent-Linking Patch (D-Pclip): The Case of a Biosensing Element.
Erika KomiyaShouhei TakamatsuDaimei MiuraKaori TsukakoshiWakako TsugawaKoji SodeKazunori IkebukuroRyutaro AsanoPublished in: Journal of the American Chemical Society (2024)
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.
Keyphrases
- circulating tumor
- cell free
- dna binding
- single molecule
- binding protein
- amino acid
- label free
- type diabetes
- healthcare
- adipose tissue
- circulating tumor cells
- risk assessment
- weight loss
- dna damage
- insulin resistance
- human health
- climate change
- ionic liquid
- dna repair
- health information
- real time pcr
- room temperature
- quantum dots
- life cycle