Selection of siRNA versus shRNA for target validation

Both siRNA and shRNA-based methods are effective in vitro as well as in vivo and easily transfect cells but the duration of action is variable. Both can be adapted to highthroughput formats.

Off-target effects of siRNA-mediated gene silencing

Off-target or non-specific effects of siRNA are a concern. Regardless of how an artificial siRNA is introduced into cells, off-target effects can occur as a result of one of three mechanisms (Cullen et al 2006):

1. Because both siRNA and shRNA contain stretches of dsRNA, they can activate nonspecific innate immune cellular responses, e.g., interferon response (see Chapter 8).

2. Transfected or expressed siRNAs can exert other non-specific effects.

3. Although mature siRNAs are designed to be fully complementary to a single mRNA transcript, they may inadvertently considerable complimentarity to other non-target mRNAs.

Off-target effects can be grouped into three main categories: (1) sequence-based effects;(2) interferon-based effects; and (3) miRNAs. Sequence-based effects are due to siRNAs targeting genes with sequences similar to the intended target of mRNA. They seem to be concentration-dependent and can be minimized by using lower RNAi concentrations and selecting sites more carefully.

A study has assessed the specificity of 10 different siRNAs corresponding to the MEN1 gene by examining the expression of two additional genes, TP53 (p53) and CDKN1A (p21), which are considered functionally unrelated to menin but are sensitive markers of cell state (Scacheri et al 2004). MEN1 RNA and corresponding protein levels were all reduced after siRNA transfection of HeLa cells, although the degree of inhibition mediated by individual siRNAs varied. Unexpectedly, the investigators observed dramatic and significant changes in protein levels of p53 and p21 that were unrelated to silencing of the target gene. The modulations in p53 and p21 levels were not abolished on titration of the siRNAs, and similar results were obtained in three other cell lines; in none of the cell lines tested did they see an effect on the protein levels of actin. These data suggest that siRNAs can induce nonspecific effects on protein levels that are siRNA sequencedependent but that these effects may be difficult to detect until genes central to a pivotal cellular response, such as p53 and p21, are studied. The authors found no evidence that activation of the dsRNA-triggered IFN-associated antiviral pathways accounts for these effects, but speculate that partial complementary sequence matches to off-target genes may result in a miRNA-like inhibition of translation.

Higly potent siRNA developed by Cenix reduce off-target effects. siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling (Chi et al 2003). A study from Abbott Laboratories has shown that when the siRNA design and transfection conditions are 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 (Semizarov et al 2003). 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.

Bioinformatic approach to off-target effects

The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. A computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, C. elegans and S. pombe (Qiu et al 2005). The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100-400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. High-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5' termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C. elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of approximately 10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.

Another computational study found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing (Du et al 2005). Mismatches formed between adenine and cytosine, in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46 (a functionally validated siRNA). This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is important for the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.

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