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In this study, a novel electrochemical biosensor was constructed for ultrasensitive and locus-specific detection of N 6 -Methyladenine (m 6 A) in DNA using double-hindered replication and nucleic acid-coated methylene blue (MB)@Zr-MOF. Based on the combination of m 6 A-impeded replication and Ag I -mediated mismatch replication, this mode could effectively stop the extension of the strand once DNA polymerase encountered m 6 A site, which specifically distinguish the m 6 A site from natural A site in DNA. Also, Zr-MOF with high porosity and negative surface potential features was carefully chose to load cationic MB, resulting a stable and robust MB@Zr-MOF electrochemical tag. As a result, the developed biosensor exhibited a wide linear range from 1 fM to 1 nM with detection limit down to 0.89 fM. Profiting from the high sensitivity and selectivity, the biosensing strategy revealed good applicability, which had been demonstrated by quantitating m 6 A DNA at specific site in biological matrix. Thus, the biosensor provides a promising platform for locus-specific m 6 A DNA analysis. Graphical Abstract

journal of nanobiotechnology

Femtomolar and locus-specific detection of N6-methyladenine in DNA by integrating double-hindered replication and nucleic acid-functionalized MB@Zr-MOF