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 CuII-responsive allosteric DNAzymes by incorporating artificial hydroxypyridone ligand-type nucleotides (H) that form a CuII-mediated base pair (H-CuII-H). We devised a new enzymatic method using a standard DNA polymerase and a ligase to prepare DNA strands containing H nucleotides. Previously reported DNAzymes were modified by introducing a H-H pair into the stem region, and the stem-loop sequences were altered so that the structure becomes catalytically inactive in the absence of CuII ions. The formation of a H-CuII-H base pair triggers intrastrand transformation from the inactive to the active structure, enabling allosteric regulation of the DNAzyme activity in response to CuII ions. The activity of the H-modified DNAzyme was reversibly switched by the addition and removal of CuII ions under isothermal conditions. Similarly, by incorporating a H-CuII-H pair into an in vitro-selected AgI-dependent DNAzyme, we have developed a DNAzyme that exhibits an AND logic-gate response to CuII and AgI 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.