Hunting Endogenous and Exogenous Radicals in DNA Cleavage Catalyzed by a Mn²⁺-Dependent Deoxyribozyme

DNAzymes, catalytically active DNA molecules, cleave DNA through metal ion cofactors (dia- and paramagnetic cofactors). A radical pathway through which cleavage occurs has been proposed for the Radical Deoxyribozyme3 (RadDz3) system. The integrative approach proposed in this work aims to identify several radical species involved in the DNA-cleaving-DNA architecture. Furthermore, we pinpoint the fine-tuning of the catalytic activity of the paramagnetic cofactors (mainly the high-spin system of Mn²⁺) combined with the role of different diamagnetic salts. The identified radical species exhibit a role both in the cleavage reaction as well as in the stabilization of the oligonucleotides' architecture. The role played by the EPR multifrequency approach is crucial in delivering consistent and robust spectroscopic parameters. These parameters can contribute to optimal fitting procedures for spectra recorded under several different conditions. This protocol proved particularly effective, despite the superstoichiometric ratio (metal/DNAzyme), typical of catalytic DNAs. The impact of different salts could also be highlighted, either in stabilizing the global architecture or affecting the catalytic activity.