Abstract
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.
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