Analysis of putative miRNA binding sites and mRNA 3' ends as targets for siRNA-mediated gene knockdown.

Tremendous efforts have been made to develop short-interfering RNA (siRNA) design algorithms that generate highly functional siRNAs for gene knockdown. Nevertheless, "difficult-to-silence" target messenger RNAs (mRNAs) still exist for which no functionally validated siRNAs are available. MicroRNA (miRNA) sites in the mRNA 3'UTR, which interact with miRNA-loaded RNA-induced silencing complex (miRISC) for posttranscriptional gene regulation, provide alternative potentially accessible sites for siRNA. To investigate this, we designed siRNAs directed against single putative miRNA sites (misiRNAs) as predicted by miRNA target databases as well as siRNAs against other regions within the 3'UTR of "difficult-to-silence" targets in this proof-of-principle study. Although their design was not fully optimized, the misiRNAs generally caused higher knockdown than previously designed siRNAs for these targets. In general, knockdown by misiRNAs targeting the miRNA seed region was specific for the target mRNA, and misiRNAs targeting 1 nt upstream of miRNA seed region were similarly potent. We also systematically screened the 3'UTR of two mRNA targets using siRNA-tiling experiments. 5' and 3' regions of the p21-activated kinase 6 (PAK6) 3'UTR were found accessible, whereas the middle portion was largely inaccessible for siRNA knockdown. In ribosomal protein S6 kinase (RPS6KB1) 3'UTR, however, only the 5' region was accessible for siRNA knockdown. Detailed analysis of 10 further "difficult-to-silence" targets revealed that siRNA accessibility at the mRNA 3' end is not a general phenomenon, at least in "difficult-to-silence" targets, as we could not detect significant knockdown by siRNAs directed against this region.

Insights into effective RNAi gained from large-scale siRNA validation screening.

Transfection of chemically synthesized short interfering RNAs (siRNAs) enables a high level of sequence-specific gene silencing. Although siRNA design algorithms have been improved in recent years, it is still necessary to prove the functionality of a given siRNA experimentally. We have functionally tested several thousand siRNAs for target genes from various gene families including kinases, phosphatases, and cancer-related genes (e.g., genes involved in apoptosis and the cell cycle). Some targets were difficult to silence above a threshold of 70% knockdown. By working with one design algorithm and a standardized validation procedure, we discovered that the level of silencing achieved was not exclusively dependent on the siRNA sequences. Here we present data showing that neither the gene expression level nor the cellular environment has a direct impact on the knockdown which can be achieved for a given target. Modifications of the experimental setting have been investigated with the aim of improving knockdown efficiencies for siRNA-target combinations that show only moderate knockdown. Use of higher siRNA concentrations did not change the overall performance of the siRNA-target combinations analyzed. Optimal knockdown at the mRNA level was usually reached 48-72 hours after transfection. Target gene-specific characteristics such as the accessibility of the corresponding target sequences to the RNAi machinery appear to have a significant influence on the knockdown observed, making certain targets easy or difficult to knock down using siRNA.