How does sirna function

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Figure 2. In this case, Argonaute recruits a member of the GW protein family. This is particularly important for off-target effects observed in RNAi experiments Seok et al. This will lead to silencing and unwanted off-target effects. Since such sequences are only 6—7 nt long, these unspecific target sites are hardly predictable and are thus very difficult to avoid. Indeed, miRNA-like off-target effects are highly problematic in large-scale RNAi screening approaches, and many hits are false positive and caused by off-target effects [e.

Thus, strategies that control for or even reduce or eliminate such off-target effects are urgently needed. In RNAi-mediated pest control, such off-target effects might not be predominantly problematic for the plant system since such a translational control system might be rather rare. However, in strategies, in which plants express si- or shRNAs that are taken up by animals and are toxic to defined species, off-target effects need to be considered.

For example, non-target animals might incorporate these RNAs as well and, although perfect complementary target RNAs are absent, the expression of partially complementary sites could be affected through the endogenous miRNA system. However, miRNA-like seed matches are difficult to predict because they statistically occur very frequently on mRNAs and not all such matches are always leading to significant knockdown effects.

A conclusive strategy to monitor such effects are whole transcriptome sequencing in case target organisms and cells are identifiable. However, molecules with strongly reduced off-target effects would be the most desirable approach. To reduce miRNA-like off-target activities, two main strategies have been developed Jackson and Linsley, ; Seok et al.

Both modifications weaken the interaction between the guide strand and the target. Since seed matches are short, such interactions are much stronger affected by this mild destabilization than siRNAs, which are typically fully complementary to their on-target.

Thus, miRNA-like off-target interactions are reduced while on-target silencing is not compromised. In addition to the modification at position 2, other modifications have also been explored [for more details, please see Seok et al. A second approach to reduce off-target effects is pooling of multiple siRNAs. It is important to notice that miRNA-like off-target effects are specific to individual sequences.

This could be achieved by administration of very low concentrations Persengiev et al. However, this would also directly affect on-target activity. Individual siRNAs within such a pool are directed against the same on-target at different positions, but each individual siRNA has a unique off-target signature.

In complex pools, concentrations of individual siRNAs are very low and thus miRNA-like off-target effects are diluted out and cannot be measured anymore. Based on these ideas, three main pooling strategies are currently used. First, in the so-called smartPools, four individual siRNAs are combined.

However, the complexity of such pools is low and thus the desired dilution effects are often not very pronounced. These pools are then applied to cell cultures and, since these highly complex pools contain hundreds of different siRNAs, sequence-specific off-targets are not observed Hannus et al. Up to 30 different siRNAs are designed and generated in vitro and such pools eliminate off-target effects even when a single siRNA with a pronounced off-target is included into the pool Hannus et al.

Chemical modifications are the preferred choice when siRNAs are used for therapeutic purposes. For drug development, single and well-defined molecule species are preferred since broad toxicological validations are required during clinical trials and final approval. SiRNA pooling strategies are preferred in individual knockdown studies for research purposes or in genome-wide RNAi screening studies. Such pools are cost-efficient and thus genome-scale libraries are available.

Plants and animals with rather primitive immune systems tolerate long dsRNA and process it to siRNAs for gene silencing. This will kill or affect growth of the pathogens. Since such complex pools are naturally generated from dsRNAs in nematodes, insects, or fungi, miRNA-like off-target activity might be neglectable, when dsRNA is applied.

In higher organisms such as mammals, the dsRNA will be fully degraded while transitioning through the digestive tract and only free nucleosides will be taken up. Thus, the administration of dsRNA to plants is an elegant and presumably very safe way of plant protection. SiRNAs are designed sequence specifically, and effects on other even highly related species could be minimized. Furthermore, since dsRNA is a natural product that is present in human diet, it might be better accepted by local communities than other plant protection strategies including the generation of genetically modified organisms GMOs or the use of conventional pesticides.

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