Overhoff Marita, Alken Martina, Far Rosel Kretschmer-Kazemi, Lemaitre Marc, Lebleu Bernard, Sczakiel Georg, Robbins Ian
Universität zu Lübeck, Institut für Molekulare Medizin, Ratzeburger Allee 160, D-23538 Lübeck, Germany.
J Mol Biol. 2005 May 13;348(4):871-81. doi: 10.1016/j.jmb.2005.03.012.
The efficiency with which small interfering RNAs (siRNAs) down-regulate specific gene expression in living cells is variable and a number of sequence-governed, biochemical parameters of the siRNA duplex have been proposed for the design of an efficient siRNA. Some of these parameters have been clearly identified to influence the assembly of the RNA-induced silencing complex (RISC), or to favour the sequence preferences of the RISC endonuclease. For other parameters, it is difficult to ascertain whether the influence is a determinant of the siRNA per se, or a determinant of the target RNA, especially its local structural characteristics. In order to gain an insight into the effects of local target structure on the biological activity of siRNA, we have used large sets of siRNAs directed against local targets of the mRNAs of ICAM-1 and survivin. Target structures were classified as accessible or inaccessible using an original, iterative computational approach and by experimental RNase H mapping. The effectiveness of siRNA was characterized by measuring the IC50 values in cell culture and the maximal extent of target suppression. Mean IC50 values were tenfold lower for accessible local target sites, with respect to inaccessible ones. Mean maximal target suppression was improved. These data illustrate that local target structure does, indeed, influence the activity of siRNA. We suggest that local target screening can significantly improve the hit rate in the design of biologically active siRNAs.
小干扰RNA（siRNA）在活细胞中下调特定基因表达的效率是可变的，并且已经提出了一些siRNA双链体的序列控制的生化参数用于设计高效的siRNA。其中一些参数已被明确确定会影响RNA诱导沉默复合体（RISC）的组装，或有利于RISC核酸内切酶的序列偏好。对于其他参数，很难确定这种影响是siRNA本身的决定因素，还是靶RNA的决定因素，尤其是其局部结构特征。为了深入了解局部靶标结构对siRNA生物学活性的影响，我们使用了大量针对ICAM-1和survivin mRNA局部靶标的siRNA。使用原始的迭代计算方法并通过实验性核糖核酸酶H作图，将靶标结构分类为可及或不可及。通过测量细胞培养中的IC50值和靶标抑制的最大程度来表征siRNA的有效性。相对于不可及的局部靶标位点，可及的局部靶标位点的平均IC50值低十倍。平均最大靶标抑制得到改善。这些数据表明，局部靶标结构确实会影响siRNA的活性。我们建议局部靶标筛选可以显著提高生物活性siRNA设计中的命中率。
