Construction of a label-free fluorescent biosensor for homogeneous detection of m 6 A eraser FTO in breast cancer tissues.

Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N ⁶ - methyladenosine (m ⁶ A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m ⁶ A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m ⁶ A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3'-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10 ^-10  mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)