Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents - A comparative analysis
Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents - A comparative analysis
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DOI：
10.1074/jbc.m210326200
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发表时间：
2003-02-28
影响因子：
4.8
通讯作者：
Baker, BF
中科院分区：
文献类型：
--
作者：
Vickers, TA;Koo, S;Baker, BF
RNA干扰可以被认为是一种反义作用机制，它利用双链核糖核酸酶来促进目标RNA的水解。我们在人类细胞中进行了一项优化的反义寡核苷酸的比较研究，这些寡核苷酸是通过RNA干扰机制工作的，而寡核苷酸是通过RNase H依赖的机制工作的。对优化的RNaseH依赖寡核苷酸和小干扰RNA(SiRNA)寡核苷酸双链的效价、最大效力、作用持续时间和序列特异性进行了评估，发现它们具有可比性。碱基错配对活性的影响在siRNA寡核苷酸和RNaseH依赖的寡核苷酸中都是位置依赖的。此外，我们确定了siRNA寡核苷酸和RNaseH依赖的寡核苷酸的活性都受到目标mRNA的二级结构的影响。为了确定靶RNA上被识别为对RNase H介导的降解敏感的位置是否与siRNA靶点一致，我们评估了旨在与RNase H依赖的寡核苷酸结合靶RNA上相同位置的siRNAs的有效性。对设计用于与来自四个不同人类基因的RNA结合的80个siRNA寡核苷酸双链的检查表明，总体上，活性与针对相同位置设计的RNase H依赖的寡核苷酸的活性相关，尽管也有一些例外。这两种策略之间的一个主要区别是，RNaseH依赖的寡核苷酸在针对前-mRNA中的靶标时被确定为活性，而siRNAs则不是。这些结果表明，siRNA寡核苷酸和RNaseH依赖的反义策略都是在细胞分析中评估基因功能的有效策略。
RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA. We have performed a comparative study of optimized antisense oligonucleotides designed to work by an RNA interference mechanism to oligonucleotides designed to work by an RNase H-dependent mechanism in human cells. The potency, maximal effectiveness, duration of action, and sequence specificity of optimized RNase H-dependent oligonucleotides and small interfering RNA (siRNA) oligonucleotide duplexes were evaluated and found to be comparable. Effects of base mismatches on activity were determined to be position-dependent for both siRNA oligonucleotides and RNase H-dependent oligonucleotides. In addition, we determined that the activity of both siRNA oligonucleotides and RNase H-dependent oligonucleotides is affected by the secondary structure of the target mRNA. To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with siRNA target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides. Examination of 80 siRNA oligonucleotide duplexes designed to bind to RNA from four distinct human genes revealed that, in general, activity correlated with the activity to RNase H-dependent oligonucleotides designed to the same site, although some exceptions were noted. The one major difference between the two strategies is that RNase H-dependent oligonucleotides were determined to be active when directed against targets in the pre-mRNA, whereas siRNAs were not. These results demonstrate that siRNA oligonucleotide- and RNase H-dependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays.