Development of fluorescence turn-on assay for enzymatic activity utilizing a nucleobase specific fluorescent ligand

Abstract

Cellular DNA suffers from a wide range of damages caused by UV light and chemical mutagens. An increasing number of examples that demonstrate the correlation between DNA repairing and disease prevention have been documented. Base excision repair (BER) that protects cells from nucleotide base damage has been considered one of the major pathways to repair cellular DNA. Thus, it is of great importance to develop assays for the DNA repair enzymes which play critical roles in the cell. In this study, by using uracil-DNA glycosylase as model enzyme, a novel, label-free, fluorescent assay for the accurate determination of BER enzyme activity has been developed by employing abasic site-containing duplex DNA and a nucleobase-specific fluorescent ligand, 2-amino-5, 6, 7-trimethyl-1, 8-naphthyridine (ATMND). ATMND as an extrinsic fluorophore can be quenched in the cytosine opposite abasic site of duplex DNA by stacking interaction with the flanking nucleobases. Our assay relies on the strategy that UDG-catalyze the removal of uracil bases from duplex DNA, which leads to the releasing of ATMND. Consequently, the released ATMND emits a high fluorescence signal. Using this new strategy, UDG is successfully analyzed with a high selectivity and sensitivity. In addition, this assay shows the potential that it can be utilized as a promising strategy for the investigation of UDG inhibitors. We strongly expect that a broad range of other enzymatic assays can be devised by using this approach.