Controllable Assembly of a DNAzyme Feedback Nanowire for Single-Molecule Detection of Fat Mass and Obesity-Associated Protein in Clinical Breast Cancer Samples

Fat mass and obesity-associated protein (FTO) is a key <i>N</i>⁶-methyladenine (m⁶A) demethylase with crucial roles in various cellular pathways, and its dysregulation is implicated in diverse diseases, including obesity and cancers. Herein, we integrate a terminal deoxynucleotidyl transferase (TdT)-mediated DNAzyme nanowire with a DNA-gold nanoparticle (AuNP) nanoprobe to construct a positive-feedback single-molecule nanosensor for the highly sensitive profiling of FTO in clinical breast tissues. In this assay, FTO can specifically remove the m⁶A modification from the substrate probe, initiating the DpnII-catalyzed cleavage reaction to produce two DNA fragments with 3'-hydroxyl termini. TdT recognizes the 3'-OH termini of DNA to catalyze template-free extension by polymerizing dTTPs, generating elongated polythymidine (poly-T) tails. The synthetic poly-T tails can hybridize with split DNAzyme sequences, inducing the assembly of long active DNAzyme nanowires that can catalyze cyclic cleavage of reporter probes on AuNPs. Upon the addition of T4 polynucleotide kinase (PNK), a feedback network is established via multicycle extension-hybridization-cleavage to induce the exponential accumulation of Cy5 molecules for the quantification of FTO. This positive-feedback nanosensor can achieve sensitive sensing of low-abundance FTO with a detection limit of 3.41 fM, effective screening of FTO inhibitors, and accurate quantification of intracellular FTO levels with single-cell sensitivity. Moreover, it allows for differentiation of FTO levels between breast cancer patient tissues and their healthy counterparts, with great potential in biological study, drug discovery, and disease diagnostics.