Cleaving DNA with DNA: cooperative tuning of structure and reactivity driven by copper ions

Abstract

Abstract/SummaryA copper-dependent self-cleaving DNA (DNAzyme or dexoxyribozyme) previously isolated byin vitroselection has been analyzed by a combination of Molecular Dynamics simulations and advanced EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction at unprecedented resolution. The minimized 46-nucleotide deoxyribozyme forms duplex and triplex substructures that flank a highly conserved catalytic core. The self-cleaving construct forms a bimolecular complex that has a distinct substrate and enzyme domains. Cleavage of the substrate is directed at one of two adjacent nucleotides and proceedsviaan oxidative cleavage mechanism that is unique to the position cleaved. The use of isotopologues of nucleotides allowed us to provide atomic resolution for the copper-substrate complex. The spectroscopic analysis overcomes the major drawbacks related to the ‘metal-soup’ scenario, also known as ‘super-stoichiometric’ ratios of cofactorsversussubstrate, conventionally required for the cleavage reaction within those nucleic acids-based enzymes. Our results pave the way for analysis on mixtures where metals/lanthanides are used as cofactors, having demonstrated that our approach may reach resolution of single nucleotide and beyond. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic option towards the applicability of spectroscopic studies, bothin vitroandin vivomatrices.