A high-throughput screen for detection of compound-dependent phosphodiester bond cleavage at abasic sites.
We have employed a DNA molecular beacon with a real abasic site, namely a 2-deoxyribose, in a fluorescent high-throughput assay to identify artificial nucleases that cleave at abasic sites. We screened a 1280 compound chemical library and identified a compound that functions as an artificial nuclease. We validated a key structure-activity relationship necessary for abasic site cleavage using available analogs of the identified artificial nuclease. We also addressed the activity of the identified compound with dose titrations in the absence and presence of a source of non-specific DNA. Finally, we characterized the phosphodiester backbone cleavage at the abasic site using denaturing gel electrophoresis. This study provides a useful template for researchers seeking to rapidly identify new artificial nucleases.
Département de Chimie Moléculaire (DCM), Laboratoire Ingénierie et Interactions BioMoléculaires (I2BM), UMR-5250, ICMG FR-2607, CNRS, Université Grenoble Alpes (UGA), 570 Rue de la Chimie, BP-53, 38041 Grenoble Cedex 9, France. Electronic address: rideout.marc@gmail.com.
Marc C
MC
Rideout
Département de Chimie Moléculaire (DCM), Laboratoire Ingénierie et Interactions BioMoléculaires (I2BM), UMR-5250, ICMG FR-2607, CNRS, Université Grenoble Alpes (UGA), 570 Rue de la Chimie, BP-53, 38041 Grenoble Cedex 9, France.
Benjamin
B
Liet
Institut des Nanosciences & Cryogénie (INAC), SPrAM - UMR 5819 CEA/CNRS/Université Grenoble Alpes, Commissariat à l'Energie Atomique, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France.
Didier
D
Gasparutto
Département de Chimie Moléculaire (DCM), Laboratoire Ingénierie et Interactions BioMoléculaires (I2BM), UMR-5250, ICMG FR-2607, CNRS, Université Grenoble Alpes (UGA), 570 Rue de la Chimie, BP-53, 38041 Grenoble Cedex 9, France. Electronic address: nathalie.berthet@ujf-grenoble.fr.
Nathalie
N
Berthet
D004247:DNA / Q000737:chemistry*; D003851:Deoxyribonucleases / Q000737:chemistry*; D003855:Deoxyribose / Q000737:chemistry*
D003855:Deoxyribose; D004247:DNA; D003851:Deoxyribonucleases
Abasic site; Artificial nuclease; DNA damage; Elimination reactions; High-throughput screen