Allosteric Regulation of DNAzyme Activities through Intrastrand Transformation Induced by Cu(II)-Mediated Artificial Base Pairing.

Allosteric regulation is gaining increasing attention as a basis for the production of stimuli-responsive materials in many research areas including DNA nanotechnology. We expected that metal-mediated artificial base pairs, consisting of ligand-type nucleotides and a bridging metal ion, could serve as allosteric units that regulate the function of DNA molecules. In this study, we established a rational design strategy for developing Cu-responsive allosteric DNAzymes by incorporating artificial hydroxypyridone ligand-type nucleotides () that form a Cu-mediated base pair (-Cu-). We devised a new enzymatic method using a standard DNA polymerase and a ligase to prepare DNA strands containing nucleotides. Previously reported DNAzymes were modified by introducing a - pair into the stem region, and the stem-loop sequences were altered so that the structure becomes catalytically inactive in the absence of Cu ions. The formation of a -Cu- base pair triggers intrastrand transformation from the inactive to the active structure, enabling allosteric regulation of the DNAzyme activity in response to Cu ions. The activity of the -modified DNAzyme was reversibly switched by the addition and removal of Cu ions under isothermal conditions. Similarly, by incorporating a -Cu- pair into an in vitro-selected Ag-dependent DNAzyme, we have developed a DNAzyme that exhibits an AND logic-gate response to Cu and Ag ions. The rational design strategy and the easy enzymatic synthetic method presented here provide a versatile way to develop a variety of metal-responsive allosteric DNA materials, including molecular machines and logic circuits, based on metal-mediated artificial base pairing.