Sensitive detection of fat mass and obesity-associated protein based on terminal deoxynucleotidyl transferase-mediated signal amplification

Abstract

Fat mass and obesity-associated protein (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) motif in ssDNA or RNA, which plays a crucial role in maintaining the relative stability of m6A modification. Meanwhile, dysregulation of FTO expression can lead to cancers and cardiovascular diseases. So quantitative analysis of FTO is of great significance for the research related with its biological functions and application in disease diagnosis. Herein, we propose a highly sensitive biosensor for FTO detection based on terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification. To be specific, FTO can specifically demethylate the substrate probe, and the emerging double strands can be cleaved by DpnII which is a methylation-sensitive restriction endonuclease. The polymerase TdT in turn recognizes the free 3′OH and initiates polymerization to produce a mass of G-quadruplexes. Thioflavin T (ThT) can then be embedded into G-quadruplexes, producing a strong fluorescence. Experimental results manifest that the fluorescence intensity is positively correlated with the abundance of FTO. Moreover, this biosensor also has good specificity and anti-interference, and the limit of detection (LOD) is down to 30.9 fM. Therefore, our biosensor can provide an efficient strategy for FTO analysis without labeling and separation, showing great potential in clinical application.