Liu C, Cheng R, Sun L Q, Tien P
Department of Molecular Virology and Bioengineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, People's Republic of China.
Biochem Biophys Res Commun. 2001 Jun 22;284(4):1077-82. doi: 10.1006/bbrc.2001.5077.
Human platelet-type 12-lipoxygenase (12-LOX) and its metabolites play a crucial role in tumor angiogenesis. A "10-23" deoxyribozyme (DNAzyme) and its phosphorothioate-modified version were designed and synthesized against the 12-LOX mRNA. Both DNAzymes were able to cleave their substrate efficiently in a time- and concentration-dependent manner in vitro. Under a multiple turnover condition, both performed well at 37 degrees C, showing the k(cat) of 1 and 0.26 min(-1), respectively. The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro. In a cell culture system, transfection of the DNAzymes into HEL cells resulted in a significant down-regulation of the 12-LOX mRNA. Furthermore, the cell extracts from the DNAzyme-transfected cells exhibited a marked reduction in the 12-LOX enzyme activity. The present results indicated the potential use of DNAzyme technology for gene function study and cancer therapy.
人血小板型12 -脂氧合酶（12 - LOX）及其代谢产物在肿瘤血管生成中起关键作用。针对12 - LOX mRNA设计并合成了一种“10 - 23”脱氧核酶（DNAzyme）及其硫代磷酸酯修饰版本。两种DNAzyme在体外均能以时间和浓度依赖的方式有效切割其底物。在多次周转条件下，两者在37℃时均表现良好，催化常数（k(cat)）分别为1和0.26 min(-1)。DNAzyme的硫代磷酸酯修饰显著提高了其在细胞中的稳定性，且在体外动力学效率无明显损失。在细胞培养系统中，将DNAzyme转染到HEL细胞中导致12 - LOX mRNA显著下调。此外，来自DNAzyme转染细胞的细胞提取物显示12 - LOX酶活性明显降低。目前的结果表明DNAzyme技术在基因功能研究和癌症治疗中的潜在应用。
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