Design and characterization of decoy oligonucleotides containing locked nucleic acids
Design and characterization of decoy oligonucleotides containing locked nucleic acids
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DOI：
10.1093/nar/30.11.2435
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发表时间：
2002-06-01
影响因子：
14.9
通讯作者：
Magnani, M
中科院分区：
文献类型：
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作者：
Crinelli, R;Bianchi, M;Magnani, M
转染顺式元件双链寡核苷酸，被称为诱饵odn，已被报道为基因治疗提供了一类新的抗原策略的有力工具。然而，诱饵方法的主要局限性之一是细胞内核酸酶对磷酸二酯寡核苷酸的快速降解。迄今为止，已有几种DNA类似物被用于克服这一问题，但缺乏疗效和/或特异性限制了它们在体内的用途。在本文中，我们研究了构象限制性核苷酸在核转录因子κ b （nf - κ b）诱饵分子设计中的应用。从含有kappaB一致结合序列的合成双链寡核苷酸开始，我们设计了一组具有锁定核酸（LNAs）的不同程度和不同位置修饰的诱饵分子。我们的研究结果表明，在kappaB序列之外添加末端LNA碱基以生成LNA- dna -LNA共聚物足以对核酸酶酶切提供可观的保护，而不会干扰转录因子的结合。相反，在kappab结合位点的背景下插入LNA替换导致稳定性进一步提高，但导致NF-kappaB对目标序列的亲和力丧失。然而，我们的结果也表明，后者的影响显然不仅取决于内部LNA取代的程度，还取决于链的位置。这一观察结果非常重要，因为它为将带有内部LNAs的DNA-LNA双链调整为具有更好的生物稳定性和抑制效果的诱饵剂提供了证据。
Transfection of cis-element double-stranded oligonucleotides, referred to as decoy ODNs, has been reported to be a powerful tool that provides a new class of antigene strategies for gene therapy. However, one of the major limitations of the decoy approach is the rapid degradation of phosphodiester oligonucleotides by intracellular nucleases. To date, several DNA analogs have been employed to overcome this issue, but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this paper we have investigated the use of conformationally restricted nucleotides in the design of decoy molecules for nuclear transcription factor kappaB (NF-kappaB). Starting from a synthetic double-stranded oligonucleotide, containing the kappaB consensus binding sequence, we designed a panel of decoy molecules modified to various extents and at various positions with locked nucleic acids (LNAs). Our results indicate that the addition of terminal LNA bases, outside the kappaB sequence, to generate LNA-DNA-LNA co-polymers was sufficient to confer appreciable protection towards nuclease digestion, without interfering with transcription factor binding. Conversely, insertion of LNA substitutions in the context of the kappaB-binding site resulted in further increased stability, but caused a loss of affinity of NF-kappaB for the target sequence. However, our results also indicate that this latter effect was apparently dependent not only on the extent but also on strand positioning of the internal LNA substitutions. This observation is of great importance since it provides evidence for the possibility of tuning DNA-LNA duplexes with internal LNAs into decoy agents with improved features in terms of biological stability and inhibitory effect.