Morita Koji, Yamate Koji, Kurakata Shin-ichi, Abe Koji, Imanishi Takeshi, Koizumi Makoto
Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan.
Nucleic Acids Res Suppl. 2002(2):99-100. doi: 10.1093/nass/2.1.99.
We studied the properties of 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) oligonucleotides as antisense molecules. Compared to a phosphorothioate (PS) DNA and RNA heteroduplex, a duplex of an ENA/PS/ENA gapmer with RNA was a more effective substrate for RNase H-mediated cleavage. We designed ENA antisense oligonucleotides (AON) targeting human vascular endothelial growth factor (VEGF) mRNA. ENA AON with a cationic polymer considerably down-regulated VEGF mRNA expression, while no down-regulation by PS AON was observed. We also investigated the influence on the gene expression of genes other than VEGF by a gene database homology search and RT-PCR analysis. This method turned out to be an efficient approach to search for non-target genes sequence-specifically down-regulated by AON. In rat plasma, an ENA/PS/ENA gapmer was very stable after 24 hr, while a bridged nucleic acid (BNA) or locked nucleic acid (LNA) oligonucleotide and a PS oligonucleotide were half degraded in 4 hr. The high stability of ENA oligonucleotides in plasma would make ENA oligonucleotides ideal for in vivo studies.
我们研究了2'-O,4'-C-乙烯桥连核酸（ENA）寡核苷酸作为反义分子的特性。与硫代磷酸酯（PS）DNA和RNA异源双链体相比，ENA/PS/ENA间隙嵌合体与RNA的双链体是核糖核酸酶H介导切割的更有效底物。我们设计了靶向人血管内皮生长因子（VEGF）mRNA的ENA反义寡核苷酸（AON）。带有阳离子聚合物的ENA AON显著下调了VEGF mRNA的表达，而未观察到PS AON有下调作用。我们还通过基因数据库同源性搜索和逆转录-聚合酶链反应（RT-PCR）分析研究了对VEGF以外基因的基因表达的影响。结果表明，该方法是一种有效搜索被AON序列特异性下调的非靶基因的方法。在大鼠血浆中，ENA/PS/ENA间隙嵌合体在24小时后非常稳定，而桥连核酸（BNA）或锁核酸（LNA）寡核苷酸以及PS寡核苷酸在4小时内降解一半。ENA寡核苷酸在血浆中的高稳定性使其成为体内研究的理想选择。
