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首页> 外文期刊>Biochemistry >Potent gene-specific inhibitory properties of mixed-backbone antisence oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides
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Potent gene-specific inhibitory properties of mixed-backbone antisence oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides

机译：由2'-脱氧-2'-氟-D-阿拉伯糖和2'-脱氧核糖核苷酸组成的混合骨干反义寡核苷酸的有效基因特异性抑制特性

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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 H 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 comprsed 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 EC_(50) 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'-meRNA-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 H degradation of the target RNA in vitro, and suggest that this mode of inhibition is likel the most important determinant for potent antisense activity.
机译：硫代磷酸酯脱氧核糖核苷酸（PS-DNA）是使用最广泛的反义抑制剂之一。 PS-DNA表现出理想的特性，例如增强的核酸酶抗性，改善的生物利用度以及诱导RNase H介导的靶RNA降解的能力。不幸的是，PS-DNA对靶RNA的结合亲和力较低，这会影响其在反义应用中的效能。我们最近发现，由2'-脱氧-2'-氟-D-阿拉伯核糖核酸（FANA）组成的磷酸二酯连接的寡核苷酸既显示出对靶RNA的高结合亲和力，又具有引起靶RNA降解RNase H的能力[Damha等人。 （1998）J.Am.Chem.Soc。 120，12976]。在本研究中，我们评估了硫代磷酸酯连接的FANA寡核苷酸（PS-FANA）的反义活性。与它们的硫代磷酸酯连接的DNA对应物相比，完全由PS-FANA构成的寡核苷酸在指导RNA的H酶切割靶RNA方面效率较低，并且仅显示出对细胞靶标表达的弱反义抑制作用。但是，发现由PS-DNA核心核心侧翼的PS-FANA组成的混合骨干低聚物具有强大的反义活性，可抑制EC_（50）值小于5 nM的特定细胞基因表达。如靶蛋白和靶mRNA的剂量依赖性降低所表明的，这种抑制是真正的反义作用。此外，mRNA片段的出现与RNA酶H介导的mRNA靶标的裂解相一致。我们还比较了PS- [FANA-DNA-FANA]一系列PS-DNA核心大小不同的混合骨干寡聚物与相应的2'-O-甲基寡核苷酸嵌合体，即PS- [2'-meRNA-DNA- 2'-meRNA]。两种类型的寡聚物均显示出对靶RNA的非常相似的结合亲和力。然而，随着DNA核心尺寸的减小，2'-O-甲基嵌合化合物的反义能力显着减弱，而2'-氟阿拉伯糖基化合物的反义能力基本上不受影响。实际上，包含单个脱氧核糖核苷酸残基核心的PS-FANA低聚物保留了显着的反义活性。这些发现与各种嵌合反义分子在体外引发靶RNA的RNase H降解的能力完全相关，并且表明这种抑制方式很可能是有效反义活性的最重要决定因素。

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