Construction of a Single-Molecule Biosensor for Antibody-Free Detection of Locus-Specific N6-Methyladenosine in Cancer Cells and Tissues

• Published in: Analytical chemistry (Washington) 2023-03, Vol.95 (12), p.5454-5462
• Main Authors: Wang, Zi-Yue, Li, Peng, Hu, Jinping, Xu, Qinfeng, Zhang, Chun-yang
• Format: Article
• Language: English
• Subjects: Antibodies; Beads; Biosensors; Breast cancer; Cancer; Chemical compounds; Chemistry; DNA nucleotidylexotransferase; Exonuclease; Fluorescence; Fluorophores; Gene expression; Hybrids; Magnetic separation; Metabolism; N6-methyladenosine; Post-transcription; Probes; Radiolabelling; Tissues
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.3c00730

N6-Methyladenosine (m6A) has emerged as a key post-transcriptional regulator in mRNA metabolism, and its dysregulation is associated with multiple human diseases. Herein, we construct a single-molecule fluorescent biosensor for antibody-free detection of locus-specific m6A in cancer cells and tissues. A 5′-biotinylated capture probe and a 3′-hydroxylated assistant probe are designed for the recognition of specific m6A-mRNA. The m6A-sensitive endoribonuclease MazF can identify and cleave the unmethylated mRNA, and the retained intact m6A-mRNA can hybridize with assistant probes and capture probes to achieve sandwich hybrids. The sandwich hybrids are immobilized on magnetic beads (MBs) to initiate the terminal deoxynucleotidyl transferase (TdT)-assisted polymerization, facilitating the continuous incorporation of Cy5-dATP to form long Cy5-polyA tails for the production of an on-bead amplified fluorescence signal. After magnetic separation and exonuclease digestion, numerous Cy5 fluorophores are released and subsequently measured by single-molecule detection. Especially, this biosensor is implemented simply and isothermally without the involvement of either radiolabeling or m6A-specific antibody. Moreover, this biosensor shows ultrahigh sensitivity with a detection limit of 2.24 × 10–17 M, and it can discriminate a 0.01% m6A level from a large pool of coexisting counterparts. Furthermore, this biosensor can be used for monitoring cellular m6A-mRNA expression and differentiating the m6A level in the breast cancer patient tissues from that in the healthy person tissues, providing a new avenue for clinical diagnosis and epitranscriptomic research.