Ota N, Warashina M, Hirano K, Hatanaka K, Taira K
National Institute for Advanced Interdisciplinary Research, Agency of Industrial Science and Technology, MITI, Tsukuba Science City 305-8562, Japan.
Nucleic Acids Res. 1998 Jul 15;26(14):3385-91. doi: 10.1093/nar/26.14.3385.
As a part of our efforts to clarify structure-function relationships in reactions catalyzed by deoxyribozymes (DNAzymes), which were recently selected in vitro , we synthesized various chimeras and analyzed the kinetics of the corresponding cleavage reactions. We focused on the binding arms and generated helices composed of binding arms and substrates that consisted of RNA and RNA, of RNA and DNA or of DNA and DNA. As expected for the rate limiting chemical cleavage step in reactions catalyzed by DNAzymes, a linear relationship between log( k cat) and pH was observed. In all cases examined, introduction of DNA into the binding helix enhanced the rate of chemical cleavage. Comparison of CD spectra of DNAzyme. substrate complexes suggested that higher levels of B-form-like helix were associated with higher rates of cleavage of the substrate within the complex. To our surprise, the enhancement of catalytic activity that followed introduction of DNA into the binding helix (enhancement by the presence of more B-form-like helix) was very similar to that observed in the case of the hammerhead ribozymes that we had investigated previously. These data, together with other observations, strongly suggest that the reaction mechanism of metal-ion-dependent DNAzymes is almost identical to that of hammerhead ribozymes.
作为我们阐明脱氧核酶（DNA酶）催化反应中结构-功能关系工作的一部分（这些脱氧核酶是最近在体外筛选得到的），我们合成了各种嵌合体，并分析了相应切割反应的动力学。我们重点研究了结合臂，并构建了由结合臂和底物组成的螺旋结构，底物由RNA与RNA、RNA与DNA或DNA与DNA组成。正如DNA酶催化反应中限速化学切割步骤所预期的那样，观察到log(kcat)与pH之间存在线性关系。在所有检测的情况下，将DNA引入结合螺旋会提高化学切割速率。对DNA酶-底物复合物的圆二色光谱比较表明，较高水平的B型样螺旋与复合物中底物的较高切割速率相关。令我们惊讶的是，将DNA引入结合螺旋后催化活性的增强（因存在更多B型样螺旋而增强）与我们之前研究的锤头状核酶的情况非常相似。这些数据，连同其他观察结果，有力地表明金属离子依赖性DNA酶的反应机制与锤头状核酶几乎相同。
