A Schematic representation of human Ago2. C Detailed view of catalytic center of the Ago2 structure from panel B. Plant Argonaute proteins are functionally diverse and are involved in various different gene silencing processes Carbonell, Generally, through their small RNA partner, plant Ago proteins are also involved in antiviral or bacterial defense mechanisms as well as responses to herbivore attack Pradhan et al.
Furthermore, in plants and also in some animal species, particularly nematodes such as Caenorhabditis elegans , siRNAs can be actively transported between cells and tissues [see, for example, Das et al. This process has been studied in C. Within pri-miRNAs, the miRNA strand itself is embedded in the stem of a local hairpin and the microprocessor cleaves the hairpin at the base of the stem.
The resulting hairpin structure, referred to as miRNA precursor pre-miRNA , is exported to the cytoplasm by the export receptor Exportin-5 in animals Bohnsack et al.
In the cytoplasm, Dicer binds to the end of the hairpin and cleaves off an approx. This mechanism is predominant in plants Song et al. In animals, however, target RNA binding as well as the mechanism of gene silencing is different. Nucleotides 2—8 of the miRNA represent the seed sequence, which is generally fully complementary to the target site while the remaining sequence is often only partially paired Rajewsky and Socci, This incomplete pairing prevents Slicer-mediated cleavage as it is observed also in siRNA-guided knockdown studies.
Instead, Argonaute proteins recruit a member of the GW protein family, which coordinates the following steps in miRNA-guided gene silencing Behm-Ansmant et al. Since GW proteins are not conserved in plants, the extent of this type of miRNA action remains to be further investigated in plants Song et al. 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.
GM structured and wrote the text. JN wrote the text and designed figures. Both authors contributed to the article and approved the submitted version. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Bartel, D. MicroRNAs: target recognition and regulatory functions. Cell , — Baulcombe, D. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol. Behm-Ansmant, I. Genes Dev. Bernstein, E. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature , — Birmingham, A. Methods 3, — Bohnsack, M.
Rna 10, — Bologna, N. In contrast, siRNA is proposed to moderate its own amplification in plants and certain animal species , such as C. Evolutionary research and studies of gene expression - specifically, how evolutionary changes in gene-regulatory networks affect phenotypic changes in an organism - have given scientists an idea of the role of miRNA in cell differentiation.
Although, as of yet, there is little evidence of the extent of miRNAs' involvement in cell differentiation, the current theory is that they function to reinforce more powerful factors that control developmental processes, particularly because many transcription factors are highly conserved between distant species while miRNA is not found in some species, such as budding yeast.
Evolutionary studies also indicate that humans alone might have over 1, species-specific or primate-specific miRNAs, each of which can bind to hundreds of different mRNA strands. However, in animals, miRNA-mediated control of gene expression is often relatively weak compared to repression by transcription factors. To what extent, then, do we depend on miRNAs to control gene expression that we need to have so many? Some theories are that, over time, new miRNAs were acquired in sync with the development of new tissue types and organs.
Additional roles that miRNA and siRNA have in gene expression involve control of the inheritance of epigenetic modifications during cell division Kloc et al.
Both rde-1 and rde-4 are genes known to be required for RNA interference in the nematode. In studies, cells derived from rde-1 and rde-4 null mutants of C. The virus could be tracked because it had been genetically engineered to express green fluorescent protein GFP , a frequently used biological protein marker.
Lower levels of fluorescence were observed in mutant cells with an enhanced RNAi response Figure 2. Small, noncoding RNAs have proven to be valuable tools for studying the roles of specific proteins in the cell. When certain sequences are used to target specific genes, thus shutting off expression of the protein product, the effects of the deficiencies on the body can be observed. This approach is being used to study the effects of abnormal RNAi expression on fetal development.
Medical researchers are also studying ways to control expression of different proteins linked to various diseases by injecting manufactured dsRNA or antisense siRNA strands into cells Whalley, However, manipulating these different forms of RNA to effectively reduce gene expression is not always so easy. Investigators have suggested that there are at least eight different steps to the algorithm for designing the most effective RNAi molecules to use in order to reduce expression Reynolds et al.
Interestingly, many of the elements that need to be considered for optimization are a direct reflection of what we know about how RNAi works—including recognition and degradation of the target mRNA and interaction between the siRNA and RISC. Information provided by studies such as these may lead to the development of drugs to treat the inappropriate expression of certain genes, or perhaps to development of RNA-injection therapies for commercially important plants and for human and animal diseases.
Already, efforts are underway to use small, noncoding RNAs for treatment of a wide array of diseases including cancer , heart disease, and various infectious diseases Boyd, For example, a number of studies have indicated that small RNAs can act as tumor suppressors in the treatment of cancer. However, there is also evidence that some miRNAs can act as oncogenes Boyd, It is clear that there is still a lot to learn about the hundreds of small RNAs in our bodies and what roles they play in gene expression.
Bernstein, E. Dicer is essential for mouse development Nature Genetics 35 , — doi Boyd, S. Everything you wanted to know about small RNA but were afraid to ask. Laboratory Investigation 88 , — doi Buchan, J.
The two faces of miRNA. Science , — doi Chen, K. The evolution of gene regulation by transcription factors and microRNAs. Nature Reviews Genetics 8 , 93— doi Heinrichs, A.
Nature Reviews Molecular Cell Biology 5 , doi Kloc, A. Current Biology 18 , Lee, R. The C. Cell 75 , — doi Reynolds, A. Rational design for RNA interference.
Nature Biotechnology 22 , — doi Sadava, D. Freeman, Sijen, T. Cell , — doi Whalley, K. Nature Reviews Genetics 7 , doi Wilkins, C. Note: The primary considerations in selecting a delivery method for siRNA are the suitability of the method to the cells and the assay requirements for duration of silencing.
The success of RNAi experiments depends on the efficiency of gene knockdown. Early work on siRNA design established conventional guidelines for siRNA structural attributes that led to reasonable functional knockdown in specific cases [1]. The properties of potent siRNAs were further refined by performing large-scale functional studies that defined thermodynamic and sequence-based rules for rational siRNA design [2].
These design algorithms greatly improved the reliability of identifying potent siRNA sequences. The Dharmacon SMARTselection algorithm was the first comprehensive rational design strategy applied to commercial collections.
Although the sequence complementarity-based mechanism underlying RNAi allows for target-specific gene knockdown, the same mechanism can result in unintended knockdown of genes not being directly targeted. Several strategies have been developed to mitigate these so-called "off-target" effects and ensure on-target activity.
Chemical modifications to the siRNA have been used successfully to promote preferential loading of the intended antisense guide strand into the RISC complex [3, 4] and reduce sense passenger strand loading and activity [5, 6]. Further, to reduce the risk of the siRNA guide strand seed region from causing off-target effects, design algorithms can incorporate filters that exclude high-frequency seed sequences from known mammalian microRNAs [7].
Chemical modifications or thermodynamic-based design considerations can also be applied to the siRNA seed region to discourage undesired interactions [5, 8, 9]. Finally, the strategy of pooling several independent siRNAs that target an individual gene has been shown to reduce the total number of non-specific gene targets and the frequency of off-target phenotypes while preserving potent target gene knockdown [10]. All of these strategies, when combined, work efficiently to reduce off-targeting and to achieve potent and specific silencing for a successful RNAi experiment.
Finally, in vivoRNAi has been used for target validation studies in animal disease models and has the potential to be used for therapeutic purposes where disease-causing genes are selectively targeted and suppressed [21].
Controls are an essential part of every siRNA experiment. At least three types of controls should be used in each siRNA and RNAi experiment: positive control, negative control and untreated control. A well-characterized positive control allows the researcher to ensure the delivery method is sufficient to achieve effective silencing. Negative controls help to separate sequence-specific effects from the effects of experimental conditions on cellular responses.
An untreated control establishes a useful baseline reference for cell phenotypes and gene expression levels. We offer a wide selection of predesigned siRNA product lines and formats. This quick selection guide will help to determine the best option for your particular needs. View PDF ». Dharmacon siRNA products are the result of scientific innovation in siRNA design and novel modification strategies to optimize potency, specificity and delivery.
Contact Us. View all applications. How can we help you? Need help? What is siRNA? How does siRNA work? How is siRNA delivered to a cell?
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