Targeting Insulin-like Growth Factor I with 10-23 DNAzymes: 2′-O-Methyl Modifications in the Catalytic Core Enhance mRNA Cleavage
Targeting Insulin-like Growth Factor I with 10-23 DNAzymes: 2′-O-Methyl Modifications in the Catalytic Core Enhance mRNA Cleavage
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DOI：
10.1021/bi201532q
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发表时间：
2012-03-20
期刊：
影响因子：
2.9
通讯作者：
Francois, Jean-Christophe
中科院分区：
文献类型：
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作者：
Fokina, Alesya A.;Meschaninova, Mariya I.;Francois, Jean-Christophe
胰岛素样生长因子 I (IGF-I) 及其同源受体 (IGF-1R) 有助于正常细胞功能和肿瘤发生。 IGF-I 信号传导在肿瘤生长中的作用已通过基于核酸的策略在体内得到证实。在这里，我们设计了第一批针对 IGF-I mRNA 的 10-23 个 DNAzyme。与需要蛋白质催化的反义方法和 RNA 干扰不同，DNAzyme 催化无蛋白质的 RNA 切割。我们鉴定了靶序列并测量了不同设计的 DNAzyme 对短合成 RNA 靶标和体外转录的 IGF-I mRNA 的催化特性。然后将最有效的切割器转染到细胞中，并使用报告基因测定来分析它们的抑制效果。我们发现增加 DNAzyme 侧翼序列的大小和用 2'-O-甲基残基修饰末端可以提高靶 RNA 的切割率。在非必需位置用六个 2'-O-甲基核糖核苷酸修饰催化环会增加或降低催化效率，具体取决于 mRNA 靶位点。在细胞中，由于 IGF-I mRNA 序列的特异性识别和切割，具有 2'-O-甲基修饰催化核心和侧翼序列的 DNAzyme 能够抑制报告基因活性。具有无活性催化核心的突变脱氧核糖核酸酶无法阻断报告基因表达，这表明10-23脱氧核糖核酸酶的RNA切割能力有助于抑制机制。我们的结果表明，具有高催化效率的核酸酶抗性 2'-O-甲基修饰 DNAzyme 可用于抑制细胞中 IGF-I 基因功能。
Insulin-like growth factor I (IGF-I) and its cognate receptor (IGF-1R) contribute to normal cell function and to tumorigenesis. The role of IGF-I signaling in tumor growth has been demonstrated in vivo using nucleic acid-based strategies. Here, we designed the first 10-23 DNAzymes directed against IGF-I mRNA. Unlike antisense approaches and RNA interference that require protein catalysis, DNAzymes catalyze protein-free RNA cleavage. We identified target sequences and measured catalytic properties of differently designed DNAzymes on short synthetic RNA targets and on in vitro transcribed IGF-I mRNA. The most efficient cleavers were then transfected into cells, and their inhibitory effect was analyzed using reporter gene assays. We found that increasing the size of DNAzyme flanking sequences and modifications of the termini with 2'-O-methyl residues improved cleavage rates of target RNAs. Modification of the catalytic loop with six 2'-O-methyl ribonucleotides at nonessential positions increased or decreased catalytic efficiency depending on the mRNA target site. In cells, DNAzymes with 2'-O-methyl-modified catalytic cores and flanking sequences were able to inhibit reporter gene activity because of specific recognition and cleavage of IGF-I mRNA sequences. Mutant DNAzymes with inactive catalytic cores were unable to block reporter gene expression, demonstrating that the RNA cleaving ability of 10-23 DNAzymes contributed to inhibitory mechanisms. Our results show that nuclease-resistant 2'-O-methyl-modified DNAzymes with high catalytic efficiencies are useful for inhibiting IGF-I gene function in cells.