Department of Chemistry and Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada.
Xiangya School of Pharmaceutical Sciences, Central South University, 172 Tongzipo Road, Changsha, Hunan, 410013, China.
Chembiochem. 2018 May 18;19(10):1012-1017. doi: 10.1002/cbic.201800049. Epub 2018 Apr 26.
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 Ca . In this work, we showed that the EtNa DNAzyme was accelerated by freezing in water in the presence of Na . The apparent K 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 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 Ca 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.
在体外选择实验的异丙醇沉淀步骤中分离出 EtNa DNA 酶。尽管与预期的辅因子没有活性，但在几种条件下观察到其 RNA 切割活性。有 Na 时，EtNa 在约 50%乙醇中高度活跃，而在水中，有 Ca 时高度活跃。在这项工作中，我们表明 EtNa DNA 酶在水存在下冷冻时会加速。在冷冻条件下，表观 K 值达到 6.2mm Na ，比室温下在水中的 K 值紧密 20 多倍。在有 10mm Na 的情况下，EtNa 在-20°C 下冷冻 0.12 小时后具有切割速率。这种效应是 EtNa 所独有的，因为除了 Na 特异性的 NaA43 外，所有其他测试的 DNA 酶都被冷冻抑制。如果使用 Ca ，冷冻也会抑制 EtNa。我们将其归因于冰晶之间微囊中 EtNa 和 Na 的浓度，但二价金属可能会使 DNA 错误折叠。总的来说，我们系统地研究了冷冻对 DNA 酶 RNA 切割活性的影响。DNA 酶序列和金属离子种类对于确定冷冻的影响都至关重要。
