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

Abstract

In this study, a novel electrochemical biosensor was constructed for ultrasensitive and locus-specific detection of N6-Methyladenine (m6A) in DNA using double-hindered replication and nucleic acid-coated methylene blue (MB)@Zr-MOF. Based on the combination of m6A-impeded replication and AgI-mediated mismatch replication, this mode could effectively stop the extension of the strand once DNA polymerase encountered m6A site, which specifically distinguish the m6A 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 m6A DNA at specific site in biological matrix. Thus, the biosensor provides a promising platform for locus-specific m6A DNA analysis. Graphical Abstract

Silver-Coordinated Watson–Crick Pairing-Driven Three-Dimensional DNA Walker for Locus-Specific Detection of Genomic N6-Methyladenine and N4-Methylcytosine at the Single-Molecule Level

“…To assess the applicability of this nanosensor for locus-specific detection of 6mdA-DNA and 4mdC-DNA in real genomic/plasmid DNAs, we analyzed the level of 6mdA in human liver cancer cell line (HepG2 cells) and the level of 4mdC in E. coli plasmid cloning vector (pUC19 vector) (Table S2), respectively (Figure ).…”

“…The LOD is determined to be 9.80 × 10 −15 M. The sensitivity of single-molecule detection is 44.2-fold higher than that of the ensemble fluorescence detection (4.33 × 10 −13 M) (Figure S6A,B), 3 orders of magnitude higher than that of the ligation-dependent supersandwich signal amplification-based electrochemical method (4.44 × 10 −11 M), 49 and 2 orders of magnitude higher than that of the nucleic acid-functionalized methylene blue (MB)@Zr-MOF-based electrochemical method (8.90 × 10 −13 M). 50 As indicated in Figure 3B, Cy5 counts enhance with the 4mdC-DNA concentration from 1.0 × 10 −14 to 1.0 × 10 −7 M. In the logarithm scale, a linear correlation is achieved between the Cy5 counts (N) and the 4mdC-DNA concentration (C) from 1.0 × 10 −14 to 1.0 × 10 −7 M over a dynamic range of 6 orders of magnitude (inset of Figure 3B). The obtained equation is N = 286.5 + 57.1 log 10 C (R 2 = 0.9893), where N is Cy5 count, and C is 4mdC-DNA concentration (nM).…”

“…54 Analysis of 6mdA and 4mdC in Real Genomic/ Plasmid DNAs. To assess the applicability of this nanosensor for locus-specific detection of 6mdA-DNA and 4mdC-DNA in real genomic/plasmid DNAs, we analyzed the level of 6mdA in human liver cancer cell line (HepG2 cells) 50 and the level of 4mdC in E. coli plasmid cloning vector (pUC19 vector) (Table S2), 55 respectively (Figure 6). Different amounts of genomic DNA are extracted from HepG2 cells and then fragmented by dsDNA fragmentase for the cellular 6mdA-DNA assay.…”

RNA methylation‐related inhibitors: Biological basis and therapeutic potential for cancer therapy

“…Moreover, at present, N6‐methyladenine detection technology is also gradually improving. For example, for the highly sensitive and site‐specific detection of m6A, a novel electrochemical biosensor was developed using double hindered replication and nucleic acid‐coated MB @Zr‐MOF 153 . With the increased understanding of RNA methylation, aberrant expression of the relevant modifying enzymes in tumour tissues is frequently found, and this dysregulation severely affects the prognosis of cancer patients.…”

Multiarmed DNA jumper and Metal-Organic Frameworks–Functionalized Paper-based Bioplatform for Small Extracellular Vesicle–derived miRNAs Assay

“…Zr-MOFs can selectively immobilize DNA molecules owing to the strong Zr-O-P bonds formed with phosphate groups. [37][38][39] However, the low load capacities and slow responsive processes of Zr-MOFs have limited their applications. [40] Recently, graphene-based materials have been combined with MOFs to enhance their conductivities and stabilities and prevent restacking.…”