Construction of a Single-Molecule Biosensor for Antibody-Free Detection of Locus-Specific N 6 -Methyladenosine in Cancer Cells and Tissues.

N 6 -Methyladenosine (m 6 A) 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 m 6 A in cancer cells and tissues. A 5'-biotinylated capture probe and a 3'-hydroxylated assistant probe are designed for the recognition of specific m 6 A-mRNA. The m 6 A-sensitive endoribonuclease MazF can identify and cleave the unmethylated mRNA, and the retained intact m 6 A-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 m 6 A-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% m 6 A level from a large pool of coexisting counterparts. Furthermore, this biosensor can be used for monitoring cellular m 6 A-mRNA expression and differentiating the m 6 A level in the breast cancer patient tissues from that in the healthy person tissues, providing a new avenue for clinical diagnosis and epitranscriptomic research.