An allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities

A series of RNA-cleaving or DNA-cleaving DNAzymes have been obtained by in vitro selection. However, engineering an allosteric DNAzyme with dual RNA-cleaving and DNA-cleaving activities is very challenging. We used an in vitro-selected pistol-like (PL) DNAzyme as a DNA scaffold for designing a DNAzyme with dual catalytic activities. We prepared the 46-nucleotide DNAzyme with DNA-cleaving activity (PL DNAzyme), and then grafted the deoxyribonucleotide residues from an 8-17 variant DNAzyme into the region of stem-loop I and the catalytic core of the PL DNAzyme scaffold. This deoxyribonucleotide residue grafting resulted in a DNAzyme with dual RNA-cleaving and DNA-cleaving activities (DRc DNAzyme). Drc DNAzyme has properties different from those of the original PL DNAzyme, including DNA cleavage sites and the required metal ion concentration. Interestingly, the RNA substrate and RNase A can act as effectors to mediate the DNA cleavage. Our results show that RNA-cleaving and DNA-cleaving activities simultaneously coexist in DRc DNAzyme, and the DNA cleavage activity can be reversibly regulated by a conformational transition.