Cieslak Marcin, Niewiarowska Jolanta, Nawrot Magdalena, Koziolkiewicz Maria, Stec Wojciech J, Cierniewski Czeslaw S
Center for Molecular and Macromolecular Research, Polish Academy of Sciences, 90-363 Lodz, Poland.
J Biol Chem. 2002 Mar 1;277(9):6779-87. doi: 10.1074/jbc.M102325200. Epub 2001 Oct 23.
A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta(1) and beta(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in beta(1) and beta(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta1-1053 and beta3-1243, significantly inhibited expression of beta(1) and beta(3) integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, beta1-1053 and beta3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta(1) and beta(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta(1) and beta(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg(2+) specifically cleaved their substrates, synthetic beta(1) and beta(3) mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2'-O-methyl groups at both the 5' and 3' ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to beta(1) and beta(3) mRNAs with 2'-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.
开发了一种基于DNA切割脱氧核酶（DNAzyme）的新方法来控制内皮细胞中β(1)和β(3)整合素的表达。为了在mRNA中设计一个特定的切割位点，DNAzyme的侧翼结构域分别来自与β(1)和β(3) mRNA中对应于1053 - 1070和1243 - 1267序列互补的寡脱氧核苷酸。这些反义寡脱氧核苷酸的硫代磷酸酯类似物，命名为β1 - 1053和β3 - 1243，在mRNA和蛋白质合成水平上显著抑制了内皮细胞和K562细胞中β(1)和β(3)整合素亚基的表达。通过流式细胞术测量，它们还特异性降低了内皮细胞和K562细胞中相应亚基的细胞表面表达。在功能测试中，β1 - 1053和β3 - 1243分别显著降低了细胞与纤连蛋白和玻连蛋白的粘附。我们设计了针对β(1)和β(3) mRNA的DNAzyme，其包含一个15个脱氧核苷酸的催化结构域，两侧分别是β(1)和β(3) DNAzyme的8个和10个脱氧核苷酸的两个底物识别片段。两种DNAzyme在Mg(2+)存在下特异性切割其底物，即合成的β(1)和β(3) mRNA片段。尽管DNAzyme在5'和3'末端都用硫代磷酸酯和2'-O-甲基基团进行了部分修饰，显示出相似的动力学参数，但由于它们对核酸酶降解的抗性更高，因此比未修饰的DNAzyme效力显著更强。与反义寡核苷酸类似，DNAzyme消除了微血管内皮细胞在纤维蛋白和基质胶中的毛细血管管形成。总之，具有2'-O-甲基修饰的针对β(1)和β(3) mRNA的DNAzyme有潜力作为基因失活剂，最终可能提供一种在体内抑制血管生成的治疗手段。
