Year: 2026
Type: preprint
Abstract: Transitioning from quantitative abundance measurements to functional activity-based assays represents a key opportunity in DNA repair research, particularly for enzymes like MUTYH that currently lack functional assay platforms. Here, we developed a FRET-based DNA switch for real-time monitoring of MUTYH glycosylase activity. The dumbbell-like architecture incorporates an 8-oxoG: A mismatch that leads to structure switching upon MUTYH-mediated adenine excision, resulting in activity-dependent fluorescence signal change. We demonstrate that the rationally designed GC-rich DNA nanoswitch can detect both E. coli MutY and human MUTYH with high specificity, enabling real-time activity monitoring directly in bacterial free cell extracts. In addition, the assay can be easily adapted for inhibitor screening, demonstrating its potential for drug screening applications. The activity-based assay was also implemented in HeLa cells and analyzed by confocal microscopy, confirming preferential localization of the MUTYH activity within the nuclear compartment. Overall, this simple, low-cost DNA-based sensor bridges the need for functional assays by directly monitoring MUTYH glycosylase activity in complex biological environments, offering a scalable platform for future screening applications.
Source: ChemRxiv
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7223400945