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PreprintCC BY-NCChemRxiv

Activity-based Detection of MUTYH using Engineered Dumbbell DNA Switches

  • Erica Belforte1
  • Soraia Fernandes2,3
  • Andrea Celeste Di Pede1
  • Neda Bagheri1
  • Laura Beltran Chamorro1
  • Ankita Sinha1
  • Raj Paul1
  • Rosanna Mattossovich4
  • Rosa Merlo4
  • Giorgia Gambardella1
  • Frank Caruso2,3
  • Francesca Cavalieri1,2,3
2 more authors
  • Anna Valenti4
  • Alessandro Porchetta1
4 affiliations
  1. 1Department of Chemical Science and Technologies, University of Rome, Tor Vergata, Via della Ricerca Scientifica 1, Rome, Italy, 00133
  2. 2Department of Chemical and Biomolecular Engineering
  3. 3University of Melbourne
  4. 4Institute of Biosciences and BioResoures, National Research Council of Italy, Via Pietro Castellino, Naples, Italy
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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.

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Cite

BibTeXRIS
BibTeX
@misc{belforte2026activitybased,
  title = {{Activity-based Detection of MUTYH using Engineered Dumbbell DNA Switches}},
  author = {Erica Belforte and Soraia Fernandes and Andrea Celeste Di Pede and Neda Bagheri and Laura Beltran Chamorro and Ankita Sinha and Raj Paul and Rosanna Mattossovich and Rosa Merlo and Giorgia Gambardella and Frank Caruso and Francesca Cavalieri and Anna Valenti and Alessandro Porchetta},
  howpublished = {ChemRxiv},
  year = {2026},
  month = oct,
  doi = {10.26434/chemrxiv.15010973/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010973/v1},
  note = {CC BY-NC},
}
RIS
TY  - UNPB
TI  - Activity-based Detection of MUTYH using Engineered Dumbbell DNA Switches
AU  - Erica Belforte
AU  - Soraia Fernandes
AU  - Andrea Celeste Di Pede
AU  - Neda Bagheri
AU  - Laura Beltran Chamorro
AU  - Ankita Sinha
AU  - Raj Paul
AU  - Rosanna Mattossovich
AU  - Rosa Merlo
AU  - Giorgia Gambardella
AU  - Frank Caruso
AU  - Francesca Cavalieri
AU  - Anna Valenti
AU  - Alessandro Porchetta
PY  - 2026
DA  - 2026/10/23
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15010973/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010973/v1
AB  - 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.
ER  -