Two DNAzymes targeting the telomerase mRNA with large difference in Mg2+ concentration for maximal catalytic activity

The 10-23 RNA-cleaving DNAzymes require divalent metal ions, preferentially Mg(2+), for catalytic activity. For intracellular applications, it is important that a DNAzyme can perform maximal cleavage at physiological concentration of Mg(2+) (0.2-2mM). We designed DNAzymes targeting the mRNA of human telomere reverse transcriptase, two of them turned out to have large difference in Mg(2+) concentration requirement (1mM vs. 20mM) for maximal activity in vitro. When the two DNAzymes were transfected into HeLa cells, only the one requiring low Mg(2+) concentration showed inhibitory activity indicating that the in vitro property regarding Mg(2+) requirement was reserved in vivo. The cleavage of target RNA mainly involves two processes, that is hybridization of DNAzyme with substrate and cleavage of substrate in the DNAzyme-substrate duplex. To explore how the optimal Mg(2+) concentration was determined, we studied the effect of Mg(2+) on the two processes. For both DNAzymes, Mg(2+) enhanced hybridization over a range of concentration far beyond 1mM. Once the DNAzymes hybridized with their 19-mer substrates without flanking sequences, the cleavages showed little difference in Mg(2+) concentration-dependence. These facts suggest that the flanking sequences played a key role in determining the Mg(2+) concentration for maximal DNAzyme activity possibly via the formation of higher order structure in the DNAzyme-substrate duplex.