DNAzymes to beta sub(1) and beta sub(3) mRNA Down-regulate Expression of the Targeted Integrins and Inhibit Endothelial Cell Capillary Tube Formation in Fibrin and Matrigel

A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta sub(1) and beta sub(3) integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to...

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Bibliographic Details
Published in:The Journal of biological chemistry 2002-03, Vol.277 (9), p.6779-6787
Main Authors: Cieslak, M, Niewiarowska, J, Nawrot, M, Koziolkiewicz, M, Stec, W J, Cierniewski, C S
Format: Article
Language:English
Subjects:
DNAzymes
fibrin
Matrigel
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Summary:A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of beta sub(1) and beta sub(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 sub(1) and beta sub(3) mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated beta 1-1053 and beta 3-1243, significantly inhibited expression of beta sub(1) and beta sub(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, beta 1-1053 and beta 3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to beta sub(1) and beta sub(3) mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for beta sub(1) and beta sub(3) DNAzymes, respectively. Both DNAzymes in the presence of Mg super(2+) specifically cleaved their substrates, synthetic beta sub(1) and beta sub(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 sub(1) and beta sub(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.
ISSN:0021-9258