A DNA array based on clickable lesion-containing hairpin probes for multiplexed detection of base excision repair activities

Abstract

DNA is under continuous assault by environmental and endogenous reactive oxygen and alkylating species, inducing the formation of mutagenic, toxic and genome destabilizing nucleobase lesions. Due to the implications of such genetic alterations in cell death, aging, inflammation, neurodegenerative diseases and cancer, many efforts have been devoted to developing assays that aim at analyzing DNA repair activities from purified enzymes or cell extracts. The present work deals with the conception and application of a new, miniaturized and parallelized on surface-DNA biosensor to measure base excision repair (BER) activities. Such a bio-analytical tool was built by using the “click chemistry” approach to immobilize, on a glass slide, fluorescent stem-loop DNA probes, which contain a specific nucleobase lesion. The performance of this new high-throughput DNA repair analysis technology was determined by detecting uracil N-glycosylase and AP-endonuclease activities from purified enzymes or in cell extracts. The applications of this device were extended to analyze, in cell extracts, the ability of two inhibitors (Uracil glycosylase inhibitor (Ugi) and methoxyamine (MX)) to block the excision of uracil and the cleavage of AP sites, respectively. Altogether, our results show that this new fluorescent DNA microarray platform provides an easy, rapid and robust method for detecting DNA N-glycosylase and AP-endonuclease activities and evaluating the effects of BER inhibitors in a multiplexed fashion.