Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2′-O-methyl RNA, phosphorothioates and small interfering RNA
Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2′-O-methyl RNA, phosphorothioates and small interfering RNA
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DOI：
10.1093/nar/gkg409
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发表时间：
2003-06-15
影响因子：
14.9
通讯作者：
Kurreck, J
中科院分区：
文献类型：
--
作者：
Grünweller, A;Wyszko, E;Kurreck, J
锁核酸 (LNA) 和双链小干扰 RNA (siRNA) 是细胞培养和体内应用中颇具前景的新型反义分子。在这里，我们比较了 LNA-DNA-LNA gapmer 寡核苷酸和具有硫代磷酸酯和嵌合 2'-O-甲基 RNA-DNA gapmer 的 siRNA 敲低香草酸受体亚型 1 (VR1) 表达的能力。发现两侧具有 4 或 5 个 LNA 且中心具有 10 或 8 个 DNA 单体的中央延伸段的 LNA-DNA-LNA gapmer 是抑制基因表达的活性 gapmer。比较共转染研究表明，siRNA 是 VR1-绿色荧光蛋白 (GFP) 表达最有效的抑制剂。观察到特异性抑制，估计 IC50 为 0.06 nM。 LNA gapmer 被发现是最有效的单链反义寡核苷酸，IC50 为 0.4 nM，比常用的硫代磷酸酯低 175 倍（IC50 类似于 70 nM）。相比之下，2'-O-甲基修饰的寡核苷酸的效率（IC50 类似于 220 nM）比硫代磷酸酯低 3 倍。 siRNA 和嵌合 LNA-DNA 寡核苷酸的高效力使其成为针对 VR1 mRNA 的细胞培养和体内应用的有价值的候选者。
Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA-DNA-LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2'-O-methyl RNA-DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA-DNA-LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1-green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC50 of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC50 of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC50 similar to70 nM). In contrast, the efficiency of a 2'-O-methyl-modified oligonucleotide (IC50 similar to220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA-DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.