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RNAi and DsiRNA: Pathway, mechanism, and design

Read this overview of the RNA interference (RNAi) pathway. Learn about its applications and design considerations for gene silencing experiments.

RNA interference (RNAi) is a conserved pathway found in most eukaryotes where dsRNAs suppress expression of genes with complementary sequences [1, 2]. RNAi has become the experimental tool of choice for studying the effects of gene silencing.

RNAi pathway and mechanism

Long dsRNAs are degraded by Dicer into siRNAs, which are usually approximately 21 base pairs (bp) long with a central 19 bp duplex and 2-base 3′ overhangs. In mammals, Dicer processing occurs in a multi-protein complex with the TAR RNA-binding protein (TRBP). The nascent siRNA associates with Dicer, TRBP, and Argonaute 2 (Ago2) to form RISC [3]. Once in RISC, one strand of the siRNA (the passenger strand) is degraded or discarded while the other strand (the guide strand) remains to direct sequence specificity of the silencing complex. The Ago2 component of RISC is a ribonuclease that will cleave the target RNA under direction of the guide strand. A schematic overview of the pathways involved in degradative RNAi is shown in Figure 1. Once the RISC complex is activated, it can move on to target additional mRNA transcript copies. This effect amplifies gene silencing and allows the therapeutic effect to last for 3–7 days in rapidly dividing cells or several weeks in non-dividing cells [4].Pathways involved in degradative RNAi

Using RNAi as a tool

Many researchers today employ synthetic RNA duplexes as their RNAi reagents, which mimic the natural siRNAs resulting from Dicer processing of long substrate dsRNAs. These synthetic RNA duplexes are transfected into cell lines and incorporated into the Dicer pathway. Although long (several hundred bp) dsRNAs are commonly employed to trigger RNAi in C. elegans or D. melanogaster, these molecules will activate the innate immune system to trigger interferon (IFN) responses in higher organisms. However, RNAi can be performed in mammalian cells using short dsRNAs, which generally do not induce IFN responses.

Dicer-substrate RNAi technology

Traditionally, siRNAs are chemically synthesized as 21mers. Alternatively, researchers can use DsiRNAs (IDT) which are chemically synthesized 27mer RNA duplexes and have an increased RNAi potency of up to 100-fold when compared to conventional 21mer siRNAs [6]. DsiRNAs are able to effectively target some sites that 21mers are not able to silence [6].

DsiRNAs are processed by Dicer into 21mer siRNAs and designed so that cleavage results in a single, desired product. This is possible due to a novel asymmetric design where the RNA duplex has a single 2-base 3′ overhang on the antisense strand and is blunt on the other end; the blunt end is modified with DNA bases, shown in Figure 2. This design provides Dicer with a single favorable binding site that helps direct the cleavage reaction. Functional polarity is introduced by this processing event, which favors antisense strand loading into RISC. The increased potency of these reagents is thought to relate to linkage between Dicer processing and RISC loading [7]. Increased antisense loading will result in increased mRNA cleavage.

Figure 2. Design of a DsiRNA. The DsiRNA has a single 2-base 3′ overhang on the antisense strand and a blunt end modified with DNA bases (shown in lower case). This is processed by Dicer into an siRNA with symmetric 2-base 3′ overhangs on each end.

Designing siRNAs

The ability of an siRNA to silence gene expression is predominantly determined by its sequence, and not all target sites are equal [5, 8]. In addition to the actual sequence, other considerations, such as cross-hybridization and chemical modifications, can alter the effectiveness of the siRNA [5].

  • Location: The location of an siRNA within the entire target gene is less of a concern for potency than the localization of the siRNA within a particular gene exon structure. Therefore, knowledge of specific splice variants is important to determine how to most effectively target the desired isoform(s) of the gene.
  • Modifications: Chemical modification is not required for siRNA function, but certain modifications are sometimes useful. Chemical modifications can decrease the susceptibility of synthetic nucleic acids to nuclease degradation and reduce their ability to trigger an innate immune response during in vivo applications [9]. Additionally, modification can be used to help cellular uptake and also to prevent unwanted participation in miRNA pathways that create off-target effects [9]. However, chemical modification can also alter the potency of an siRNA and so should be empirically tested to ensure they are effective. siRNAs must have phosphate groups at the 5′ end in order to have activity so it is important to not block the 5′ end of the antisense strand with any modifications other than phosphate [5]. However, transfected, unmodified 5′-OH ends are rapidly phosphorylated by cellular kinases so it is not necessary to phosphorylate synthetic siRNAs [5]. RNA, rather than dTdT dinucleotide overhangs are now recommended at the 3’ ends of each strand to improve RISC loading efficiency, and modified bases are often used to increase stability [10].
  • Thermodynamic stability: The most effective siRNAs have a relatively low Tm and duplex stability (less stable, more A/U rich) toward the 5′-end of the guide strand and a relatively high Tm (more stable, more G/C rich) toward the 5′-end of passenger strand [5]. When options are limited for a particular target sequence, it may not be possible to select thermodynamically favorable regions. For these situations, it is possible to introduce mismatches (to lower Tm) or to add modified bases (to increase Tm) to the siRNA duplex to create thermodynamic asymmetry. If bases that are non-complementary to the target must be introduced, it is important that they are on the 3′-end of the passenger strand rather than on the 5’-end of the guide strand to avoid impairing the ability of the guide strand to anneal to the active target.
  • Sequence characteristics and specificity: To maintain specificity, the guide strand should not contain sequence characteristics such as homopolymeric runs (those with four or more identical nucleotides) or nine-base or greater segments of G/C bases [5]. In addition, the secondary structure of the target and the site accessibility are important factors in the activity of siRNAs [5]. A moderate to low GC content (30–52%) is typically a feature of functional siRNAs [5].

Screening for complementarity

Like targeted effects, off-target effects (OTEs) are dose dependent. Therefore, it is important to establish dose-response profiles for all siRNAs and always use the lowest concentration of siRNA that will provide adequate target knockdown. An additional measure to prevent OTE bias is to ensure that at least two, and ideally three, independent siRNAs against a target give the same result [5].

In Vivo use of siRNAs

Use of siRNAs in vivo shows great potential as both research tools and as therapeutic agents [11]. For more information on the status of RNAi in therapeutics, see the recent review by Vaishnaw et al. [12]. Before you begin RNAi studies in vivo, consider the following issues: site selection, compound design and chemistry, controls, route of administration, and use of a delivery vehicle [11]. To find the best candidates, it is very important to validate siRNA duplexes in vitro before moving on to in vivo experiments. In addition, choose more than one effective siRNA for each target to be tested in order to rule out false positive results caused by off-target effects [11].

For an example of research currently underway using RNAi in vivo to target the central nervous system, see the Your Research article, Using DsiRNA to map pain pathways in the CNS.

Tools to understand gene function

RNAi is a powerful tool for studying gene silencing and its effects. Here we have described critical design parameters, the importance of controlling for off-target effects, and considerations for in vivo gene silencing. Advancements to the technology, such as IDT DsiRNAs, have led to even greater advancements in the potency of RNA interference. Such tools will continue to provide the means to study the role specific genes play through the effects of silencing them. For more information on DsiRNAs from IDT, visit the Custom DsiRNA product page.



Double-stranded RNA


Dicer-substrate RNAs; chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional 21mer siRNAs.

Guide strand

The antisense strand of the siRNA; will be incorporated in RISC

Passenger strand

The sense strand of the siRNA; will be degraded


RNA-Induced Silencing Complex


Small interfering RNA


An endoribonuclease that degrades long dsRNAs into small, effector molecules called siRNAs


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  2. Meister G, Tuschl T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature, 431(7006): 343–349.
  3. Chendrimada TP, Gregory RI, et al. (2005) TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature, 436(7051): 740–744.
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  10. Strapps WR, Pickering V, et al. (2010) The siRNA sequence and guide strand overhangs are determinants of in vivo duration of silencing. Nucleic Acids Res, 38: 4788–4797.
  11. Rettig GR, Behlke MA. (2012) Progress towards in vivo use of siRNAs-II. Mol Ther, 20(3): 483–512;
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Published Sep 12, 2011
Revised/updated Jan 21, 2017