Two DNAzymes targeting the telomerase mRNA with large difference in Mg 2+ concentration for maximal catalytic activity

Abstract

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+ (0.2–2 mM). 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 (1 mM vs. 20 mM) 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 1 mM. 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.