Mismatch discrimination and efficient photomodulation with split 10–23 DNAzymes

DNA enzymes (DNAzymes) that catalyze the degradation of complementary RNA molecules have been investigated for many biochemical and sensing applications. Here, we investigated a 10–23 DNAzyme that has been shown previously to possess cellular activity. We determined that it has very low Mg2+ ion dependence, with DNAzyme activity observed at [Mg2+] = 0.01 mM. This metal ion dependence is much lower than is typical for DNAzymes studied to date, and suggests that DNAzymes may be engineered for many additional biological applications. Recently, we demonstrated that this 10–23 DNAzyme can be divided into two parts, which assemble into an active oligonucleotide complex. We investigated in more detail the functionality of the split 10–23 DNAzyme and found that dividing the 15-nucleotide catalytic loop after the 7th or 8th base maximized its activity. The split DNAzymes required higher metal ion concentrations ([Mg2+] = 5 mM), and as we anticipated due to their lower hybridization activity, the split enzymes had the advantage of being more sensitive to single base mismatches in the DNAzyme–RNA duplex. Finally, we demonstrated facile photomodulation of split DNAzyme activity by incorporating a photocleavable biotin moiety bound to streptavidin.