An RNA-Cleaving Catalytic DNA Accelerated by Freezing.

The EtNa DNAzyme was isolated during the isopropanol precipitation step of an in vitro selection effort. Although inactive with the intended cofactor, its RNA cleavage activity was observed under a few conditions. With Na+ , EtNa was highly active in ∼50 % ethanol, whereas in water, it was highly active with Ca2+ . In this work, we showed that the EtNa DNAzyme was accelerated by freezing in water in the presence of Na+ . The apparent Kd value reached 6.2 mm Na+ under the frozen condition, over 20 times tighter than that in water at room temperature. With 10 mm Na+ , EtNa had a cleavage rate of 0.12 h-1 after freezing at -20 °C. This effect was unique to EtNa, as all other tested DNAzymes were inhibited by freezing except for the Na+ -specific NaA43. Freezing also inhibited EtNa if Ca2+ was used. We attributed this to the concentrations of EtNa and Na+ in the micropockets between ice crystals, but divalent metals might misfold DNA. Overall, we have systematically studied the effect of freezing on the RNA-cleavage activity of DNAzymes. The DNAzyme sequence and the metal ion species are both crucial to determine the effect of freezing.