Terminal Deoxynucleotidyl Transferase and Rolling Circle Amplification Induced G-triplex Formation: A Label-free Fluorescent Strategy for DNA Methyltransferase Activity Assay

Abstract

It is of great significance to sensitively detect DNA methyltransferase (MTase) activity and screen its inhibitory drugs in the field of human cancer diagnosis and disease therapeutics. In this work, based on terminal deoxynucleotidyl transferase (TdT)-induced rolling circle amplification (RCA), we construct a label-free and highly sensitive fluorescence biosensing strategy for DNA MTase activity analysis. The hairpin substrate is methylated by Dam MTase and cleaved by Dpnl to generate a DNA fragment with a free 3′-hydroxy (3′−OH) terminus that is extended by TdT to obtain a long adenine (poly-A) tail. Subsequently, the resulting poly-A work as primer triggers RCA reaction and generate lots of G-triplexes which can combine with Thioflavin T (ThT) to achieve the apparent fluorescence signal. Integrate TdT-induced RCA with G-triplex@ThT, Dam MTase can be detected down to 0.058 U/mL with a wide linear range from 0.1 U/mL to 40 U/mL. In addition, the method exhibits high selectivity to other MTases. We also successfully demonstrate this work can be used in complex matrixes assay and screen MTase inhibitors. Thus, in the area of DNA MTase-related cancer diagnosis and disease therapeutics, the developed method may be a promising tool for the detection of Dam MTase.