Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2′-deoxy-2′-fluoro-D-arabinose and 2′-deoxyribose nucleotides
Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2′-deoxy-2′-fluoro-D-arabinose and 2′-deoxyribose nucleotides
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DOI：
10.1021/bi0115075
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发表时间：
2002-03-12
期刊：
影响因子：
2.9
通讯作者：
Parniak, MA
中科院分区：
文献类型：
--
作者：
Lok, CN;Viazovkina, E;Parniak, MA
硫代脱氧核糖核苷酸(PS-DNA)是应用最广泛的反义抑制剂之一。PS-DNA具有良好的特性，如增强的核酸酶抗性，提高的生物利用度，以及诱导RNaseH介导的靶RNA降解的能力。不幸的是，PS-DNA与靶RNA的结合亲和力相对较低，这影响了其在反义应用中的效力。我们最近发现，由2‘-脱氧-2’-氟-D-阿拉伯核苷酸(Fana)组成的磷酸二酯连接的寡核苷酸既与目标RNA具有高的结合亲和力，又能够诱导目标RNA的RNase11降解[Damha等人]。(1998)J.Am化学。SoC。120、12976]。在本研究中，我们评价了硫代修饰的Fana寡核苷酸(PS-Fana)的反义活性。与硫代连接的DNA相比，完全由PS-Fana组成的寡核苷酸在引导靶RNA的RNase H切割方面的效率略低，并且仅显示出对细胞靶表达的微弱反义抑制。然而，由PS-DNA中心核心两侧的PS-Fana组成的混合骨架寡聚体被发现具有很强的反义活性，抑制特定细胞基因的表达，EC50值小于5 nM。这种抑制是真正的反义效应，如靶蛋白和靶mRNA的剂量依赖性下降所表明的那样。此外，mRNA片段的出现与RNase H介导的mRNA靶标的切割一致。我们还比较了一系列不同PS-DNA核心大小的PS-[Fana-DNA-Fana]混合骨架低聚物与相应的2‘-O-甲基寡核苷酸嵌合体，即PS[2’-Me-RNA-DNA-2‘Merna]。这两种类型的寡聚体对靶RNA的结合亲和力非常相似。然而，2‘-O-甲基嵌合化合物的反义效价随着DNA核大小的减小而显著减弱，而2’-氟阿拉伯基化合物的反义效价基本不受影响。事实上，含有单个脱氧核苷酸残基核心的PS-FANA寡聚体保留了显著的反义活性。这些发现恰好与各种嵌合反义分子在体外诱导靶RNA降解RNase11的能力相关联，并表明这种抑制模式可能是有效反义活性的最重要决定因素。
Phosphorothioate deoxyribonucleotides (PS-DNA) are among the most widely used antisense inhibitors. PS-DNA exhibits desirable properties such as enhanced nuclease resistance, improved bioavailability, and the ability to induce RNase H mediated degradation of target RNA. Unfortunately, PS-DNA possesses a relatively low binding affinity for target RNA that impacts on its potency in antisense applications. We recently showed that phosphodiester-linked oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (FANA) exhibit both high binding affinity for target RNA and the ability to elicit RNase 11 degradation of target RNA [Damha et al. (1998) J. Am. Chem. Soc. 120, 12976]. In the present study, we evaluated the antisense activity of phosphorothioate-linked FANA oligonucleotides (PS-FANA). Oligonucleotides comprised entirely of PS-FANA were somewhat less efficient in directing RNase H cleavage of target RNA as compared to their phosphorothioate-linked DNA counterparts, and showed only weak antisense inhibition of cellular target expression. However, mixed-backbone oligomers comprised of PS-FANA flanking a central core of PS-DNA were found to possess potent antisense activity, inhibiting specific cellular gene expression with EC50 values of less than 5 nM. This inhibition was a true antisense effect, as indicated by the dose-dependent decrease in both target protein and target mRNA. Furthermore, the appearance of mRNA fragments was consistent with RNase H mediated cleavage of the mRNA target. We also compared a series of PS-[FANA-DNA-FANA] mixed-backbone oligomers of varying PS-DNA core sizes with the corresponding 2'-O-methyl oligonucleotide chimeras, i.e., PS[2'-me-RNA-DNA-2'meRNA]. Both types of oligomers showed very similar binding affinities toward target RNA. However, the antisense potency of the 2'-O-methyl chimeric compounds was dramatically attenuated with decreasing DNA core size, whereas that of the 2'-fluoroarabino compounds was essentially unaffected. Indeed, a PS-FANA oligomer containing a single deoxyribonucleotide residue core retained significant antisense activity. These findings correlated exactly with the ability of the various chimeric antisense molecules to elicit RNase 11 degradation of the target RNA in vitro, and suggest that this mode of inhibition is likely the most important determinant for potent antisense activity.