Abstract: Engineering deoxyribozyme (DNAzyme) by the introduction of new recognition elements is an important approach for expanding its applications, however, this depends on the systematic characterization of the engineerable sites in the DNAzyme. In this work, we investigated the engineerable sites of an organic solvent-needing RNA-cleaving DNAzyme (RCD, named E3) previously reported by our group. We first split E3 into two parts at its variable region, and added two DNA sequences on the 5’-end of one part and 3’-end of the other part to form duplex to active the DNAzyme. By construction of the zipper-like Multiple Component DNAzyme (MNAzyme), we were able to identify 6 splitting sites that could be utilized for DNAzyme engineering. Based on this discovery, a DNAzyme system that was activated by an external DNA initiator was constructed by splitting E3 at its variable region and introducing two oligonucleotides at the split sites for the binding of the initiator. Furthermore, we identified several splitting sites on the substrate-binding arm of E3 as an alternative region for the design of MNAzyme. Application in both regions, an intricate three component DNAzyme system was designed to be activated only in the presence of the two specific initiators. All the MNAzyme systems exhibited extremely high selectivity and very low background both in 35% DMSO and 30% ethanol. Moreover, in mixing of various MNAzyme systems and all the relative initiators, the cleavage products were almost the sum of that in mixing these MNAzymes in the presence of individual initiator. These results demonstrate that E3 is an excellent scaffold for constructing responsive RCD probes in applications in organic solvents-containing scenarios.

Key words: DNAzyme; Stimuli-responsive, Organic solvent, Molecular engineering; Multiple component DNAzyme