Ratiometric fluorescent method for highly sensitive detection of DNA methyltransferase activity and inhibitor screening based on the DNA strand replacement reaction initiated by “toehold”

Abstract

DNA methylation has been generally regarded as predictive biomarkers for tumor diagnosis and potential biotarget for the treatment of tumors. Herein, we present dual-emission ratiometric fluorescent system primarily based on DNA strand replacement reaction initiated by“toehold”for accurately assay of Dam MTase activity. This new strategy involved thiolated double-loop hairpin DNA probe with a specific recognition sites for Dam MTase and DpnI endonucleases which was labeled by fluorescent dye FAM at 5′ end (DH-DNA), a DNA labeled by fluorescent dye Cy5 at 5′ end (T-DNA). The DH-DNA probe was previously attached on Au nanoparticles (Au NPs) surface at 5′ end (Au-HP-DNA), resulting in the fluoresence quencher of the FAM via extinction of Au NPs. Upon addition of Dam MTase, Au-HP-DNA will be methylated and cleaved into two fragments. One of the Au NPs-labeled DNA fragment can be served as“toehold”to hybridization with T-DNA and thus displacement of FAM-labeled DNA fragment, resulting in significantly fluoresence quencher of Cy5 and obvious increase of fluorescent signal of FAM. Benefiting from the built-in correlation that eliminates for external interferential effects and the large range of emission ratios, the protocol allows improved sensitivity and accurate assay for Dam MTase with a detection limit of 0.0036 U/mL. Furthermore, the protocol can be extended to screen suitable inhibitor of Dam. Thus, the present protocol might be help for further application in the therapeutic associated with DNA methylation.