Terminal deoxynucleotidyl transferase-activated nicking enzyme amplification reaction for specific and sensitive detection of DNA methyltransferase and polynucleotide kinase

DNA methyltransferase (MTase) and polynucleotide kinase (PNK) are both DNA-dependent enzymes that play important roles in DNA methylation and DNA repair processes, respectively. Dysregulation of their activities is associated with various human diseases. Herein, we present a specific and sensitive biosensing strategy, named terminal deoxynucleotidyl transferase (TdT)-activated nicking enzyme amplification reaction (TdT-NEAR), for their activity detection. As for MTase detection, an enclosed dumbbell-shaped oligonucleotide substrate, whose symmetric stem containing a recognition site of Dam MTase and an incomplete recognition sequence of nicking endonuclease Nt.BbvCI, was used. Typically, the substrate is methylated by Dam MTase and subsequently cleaved by Dpn I. In the presence of TdT and dGTP, poly(guanine, G) sequences are extended from the released 3'-OH ends, achieving the conversion of the incomplete Nt.BbvCI recognition sequence to an intact one. The extension products can then be used to trigger Nt.BbvCI-catalyzed cyclic cleavage of fluorophore/quencher-labelled oligonucleotide probe, giving a significantly enhanced fluorescence output. Such a sensing system can achieve sensitive and specific detection of Dam MTase with a detection limit of 0.002 U/mL. The unique working mechanism endows the sensing system with improved anti-interference capability and thus increased application potential in complex biological samples. Moreover, it was also demonstrated to work well for Dam MTase inhibitor screening and inhibitory activity evaluation, thus holding great potential in disease diagnosis and drug discovery. Using a simpler 3'-phosphorylated linear substrate and the same fluorescent probe, the TdT-NEAR strategy can be easily extended to the activity analysis of PNK, thus revealing wide application potential in bioanalysis.