Light-regulated catalysis by an RNA-cleaving deoxyribozyme
Light-regulated catalysis by an RNA-cleaving deoxyribozyme
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DOI：
10.1016/j.jmb.2004.06.060
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发表时间：
2004-08-20
影响因子：
5.6
通讯作者：
Sen, D
中科院分区：
文献类型：
--
作者：
Liu, Y;Sen, D
我们描述了光诱导开关的催化活性的小，RNA切割8-17脱氧核酶（DNAzyme），基于光化学诱导的顺反异构化偶氮苯（AZ）部分共价拴在不同的位置内的DNAzyme。先前的研究表明，反式偶氮苯能够在DNA双螺旋内舒适地堆叠，使其稳定，而顺式偶氮苯具有螺旋不稳定作用。我们设计了两类AZ修饰的8-17 DNA酶构建体，其中每个构建体中两个偶氮苯分子取代了底物结合臂（SBA）中的核苷酸;或者，在催化核心内。RNA切割的单转换动力学的测量显示，在SBA构建体E11和E13中，当用可见光（有利于反式-Az）照射反应混合物时，与紫外光（其促进顺式-Az）相比，获得高5至6倍的催化速率，这与稳定酶-底物复合物的这些构建体中的反式-Az一致。令人惊讶的是，用催化核心构建体E17获得了相反的结果，其中紫外线照射导致相对于可见光照射快五至六倍的催化活性。这种光响应核酸酶的发展可能会打开新的可能性，使用光作为激活或抑制剂在活细胞和生物体内的基因表达的控制。(C)2004爱思唯尔有限公司保留所有权利。
We describe light-induced switches for the catalytic activity of the small, RNA-cleaving 8-17 deoxyribozyme (DNAzyme), based on photochemically induced cis-trans isomerization of azobenzene (Az) moieties covalently tethered at various locations within the DNAzyme. Prior studies have shown that trans-azobenzene is able to stack comfortably within a DNA double helix, stabilizing it, while cis-azobenzene has a helix-destabilizing effect. We designed two classes of Az-modified 8-17 DNAzyme constructs, in each of which two azobenzene molecules substituted for nucleotides, either in the substrate-binding arm (SBA); or, within the catalytic core. Measurement of single-turnover kinetics for RNA cleavage revealed that in the SBA constructs Ell and E13, five- to sixfold higher catalytic rates were obtained when the reaction mixture was irradiated with visible light (favouring trans-Az) as compared to ultraviolet light (which promotes cis-Az), consistent with trans-Az in these constructs stabilizing the enzyme-substrate complex. Surprisingly, the reverse result was obtained with the catalytic core construct E17, where ultraviolet irradiation resulted in a five- to sixfold faster catalytic activity relative to visible light irradiation. The development of such light-responsive nucleic acid enzymes may open new possibilities of using light as the activating or repressing agent in the control of gene expression within living cells and organisms. (C) 2004 Elsevier Ltd. All rights reserved.