Specificity of short interfering RNA determined through gene expression signatures

Abstract

Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.DNA microarray ͉ RNA interference ͉ gene knockdown T he use of RNA interference for inhibiting gene expression represents a powerful tool for exploring gene function (1-7), identifying and validating new drug targets, and treating disease (8, 9). The process of RNA interference is mediated by doublestranded RNA, which is cleaved by the enzyme DICER into 21-to 23-nt duplexes containing a 2-nt overhang at the 3Ј end of each strand. These duplexes are incorporated into a protein complex called the RNA-induced silencing complex (RISC). Directed by the antisense strand of the duplex, RISC recognizes and cleaves the target mRNA (for recent reviews, see refs. 1 and 10-13). Although long double-stranded RNAs (Ͼ30 nt) invoke an interferon response, short interfering RNAs (siRNAs) that resemble the products produced by DICER have been reported to specifically inhibit gene expression in many different mammalian cell lines (1-7). It has been shown that even single nucleotide mismatches between the antisense strand of the siRNA and target mRNA can abolish RNA interference (14). In addition, mapping of mRNA cleavage sites has revealed no cleavage sites outside of the region of complementarity (10). However, the specificity of siRNA at the cellular level remains to be comprehensively studied.For siRNAs to be a useful tool in gene knockdown experiments, it is critical that siRNA-mediated transcriptional silencing be specific. It is not enough to simply show that a control siRNA with a scrambled nucleotide sequence fails to knock down the protein of interest or produce the same cellular phenotype. Ideally, the siRNA must not cause any effects other than those related to the knock down of the target gene. There are several types of nonspecific effects that siRNA could potentially display. In addition to the possibility for cross-hybridization of the antisense strand of the siRNA to different mRNAs, siRNAs could bind in a sequencedependent manner to various cellular proteins. I...

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“…In this respect, some studies have shown that even 11-bp matches between a siRNA and an unintended target is enough to impede that gene [10]. In general, the magnitude of this unwanted activity known as an "off-target effect" was lower than that of the on-target gene, and can be avoided by careful selection of siRNA sequences and working with the lowest effective siRNA concentrations [11].…”

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“…Some siRNAs have 'off-target' effects, which are often the result of partial homology to other transcripts 11,12 . The shRNA library was designed to avoid off-target effects by minimizing homology of shRNAs to other transcripts (see Methods).…”

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“…For some siRNA, single nucleotide mismatches have been reported to abolish activity, whereas others have reported an absence of RNAi effects only after introduction of 7 mutations (12,13). When, instead of focusing on the target gene alone, all genes are examined for siRNA effects, the picture becomes less clear (14)(15)(16). Three conflicting reports have been published thus far: two describing the absence of any interference of siRNA on non-target mRNA (14,15), whereas the other report claims cross-reaction of siRNA with approximately 0.1% of the investigated 21,000 transcripts with even only limited (seven nucleotides) or no sequence similarity (16).…”

“…When, instead of focusing on the target gene alone, all genes are examined for siRNA effects, the picture becomes less clear (14)(15)(16). Three conflicting reports have been published thus far: two describing the absence of any interference of siRNA on non-target mRNA (14,15), whereas the other report claims cross-reaction of siRNA with approximately 0.1% of the investigated 21,000 transcripts with even only limited (seven nucleotides) or no sequence similarity (16). The differences between these studies could be the result of siRNA concentration as higher concentrations have been associated with a different pharmacological profile (15).…”

“…Three conflicting reports have been published thus far: two describing the absence of any interference of siRNA on non-target mRNA (14,15), whereas the other report claims cross-reaction of siRNA with approximately 0.1% of the investigated 21,000 transcripts with even only limited (seven nucleotides) or no sequence similarity (16). The differences between these studies could be the result of siRNA concentration as higher concentrations have been associated with a different pharmacological profile (15). Yet, Jackson et al also reported off-target regulation at very low siRNA concentrations albeit that these effects were far less pronounced than for the higher concentrations (16).…”