Opalinska J B, Kalota A, Gifford Lida K, Lu Ponzy, Jen Kuang-Yu, Pradeepkumar P I, Barman J, Kim T K, Swider C R, Chattopadhyaya J, Gewirtz A M
Division of Hematology/Oncology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Nucleic Acids Res. 2004 Oct 28;32(19):5791-9. doi: 10.1093/nar/gkh893. Print 2004.
Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.
将具有新型碱基约束氧杂环丁烷（OXE）修饰（氧杂环丁烷，1-(1',3'-O-脱水-β-D-拟呋喃核糖核苷））的核苷掺入反义（AS）寡脱氧核糖核苷酸（ODN）中应能大大提高这些分子的基因沉默效率。这是因为OXE修饰的碱基为天然骨架ODN提供核酸酶保护，能赋予与RNA-RNA杂交体预测值相似的熔解温度（Tm）值，不仅允许而且加速核糖核酸酶H介导的催化活性。我们通过直接比较OXE和硫代磷酸酯（PS）ODN靶向c-myb基因表达的能力，在活细胞中测试了这一假设。这些ODN靶向c-myb mRNA内的两个不同位点。一个位点是任意选择的。另一个是基于与自猝灭报告分子（SQRM）进行物理作图后预测的杂交可及性做出的“合理”选择。OXE和PS ODN均能同等程度地降低Myb mRNA和蛋白质水平，但后者递送至细胞的效率约高六倍，这表明OXE修饰的ODN在摩尔基础上更有效。合理靶向的分子比靶向任意选择的mRNA序列的分子表现出更高的沉默效率。我们得出结论，合理靶向的OXE修饰ODN可有效地作为基因沉默剂发挥作用，并推测它们将被证明对治疗目的有用。
