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Evidence of a General Acid–Base Catalysis Mechanism in the 8–17 DNAzyme
Biochemistry ( IF 2.9 ) Pub Date : 2018-02-01 00:00:00 , DOI: 10.1021/acs.biochem.7b01096
Marjorie Cepeda-Plaza 1 , Claire E McGhee 2 , Yi Lu 2
Affiliation  

DNAzymes are catalytic DNA molecules that can perform a variety of reactions. Although advances have been made in obtaining DNAzymes via in vitro selection and many of them have been developed into sensors and imaging agents for metal ions, bacteria, and other molecules, the structural features responsible for these enzymatic reactions are still not well understood. Previous studies of the 8–17 DNAzyme have suggested conserved guanines close to the phosphodiester transfer site may play a role in the catalytic reaction. To identify the specific guanine and functional group of the guanine responsible for the reaction, we herein report the effects of replacing G1.1 and G14 (G; pKa,N1 = 9.4) with analogues with a different pKa at the N1 position, such as inosine (G14I; pKa,N1 = 8.7), 2,6-diaminopurine (G14diAP; pKa,N1 = 5.6), and 2-aminopurine (G14AP; pKa,N1 = 3.8) on pH-dependent reaction rates. A comparison of the pH dependence of the reaction rates of these DNAzymes demonstrated that G14 in the bulge loop next to the cleavage site, is involved in proton transfer at the catalytic site. In contrast, we did not find any evidence of G1.1 being involved in acid-base catalysis. These results support general acid–base catalysis as a feasible strategy used in DNA catalysis, as in RNA and protein enzymes.

中文翻译:

8-17 DNAzyme 中通用酸碱催化机制的证据

DNAzyme 是具有催化作用的 DNA 分子,可以进行多种反应。尽管通过体外选择获得脱氧核糖核酸酶已经取得了进展,并且其中许多脱氧核糖核酸酶已被开发成金属离子、细菌和其他分子的传感器和成像剂,但导致这些酶促反应的结构特征仍然没有得到很好的了解。先前对 8-17 DNAzyme 的研究表明,靠近磷酸二酯转移位点的保守鸟嘌呤可能在催化反应中发挥作用。为了确定负责该反应的特定鸟嘌呤和鸟嘌呤官能团,我们在此报告了用N1 处具有不同 pKa 的类似物替换 G1.1 和 G14 (G; pKa,N1 = 9.4 )效果位置,例如肌苷 (G14I; p Ka ,N1 = 8.7)、2,6-二氨基嘌呤 (G14diAP; p Ka ,N1 = 5.6) 和 2-氨基嘌呤 (G14AP; p Ka ,N1 = 3.8) pH 依赖性反应速率。对这些 DNAzyme 反应速率的 pH 依赖性的比较表明,靠近裂解位点的凸环中的 G14 参与催化位点的质子转移。相反,我们没有发现任何G1.1参与酸碱催化的证据。这些结果支持一般酸碱催化作为 DNA 催化(如 RNA 和蛋白酶)中使用的可行策略。
更新日期:2018-02-01
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