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RNAi and DsiRNA: pathway, mechanism and design

4th March 2013


Since its discovery, scientists have co-opted the RNAi mechanism as an experimental tool for studying the effects of gene silencing both in vitro and in vivo. Jaime Sabel and Hans Packer report.

RNA interference (RNAi) is a conserved pathway found in most eukaryotes where short, dsRNAs suppress expression of genes with complementary sequences1-2.

In mammalian cells, RNAi occurs when long dsRNA molecules are processed by Dicer into siRNAs, which are 21 bp long dsRNAs, with a central 19 bp duplex, and characteristic 2-base, 3' overhangs. Dicer processing occurs in a multi-protein complex that includes the TAR RNA-binding protein (TRBP). The nascent siRNA associates with Dicer, TRBP, and Argonaute 2 (Ago2) to form RISC3.

Once incorporated into 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 cleaves target RNAs under direction of the guide strand.

Once the RISC complex is activated by an siRNA, it can target numerous mRNA transcript copies. This multiple targeting by a single siRNA molecule amplifies gene silencing, allowing the effects to persist for 3-7 days in rapidly dividing cells and up to several weeks in non-dividing cells4.

Synthetic RNA Duplexes as siRNA Reagents. Synthetic RNA duplexes that mimic natural siRNAs allow researchers to take advantage of the RNAi mechanism in cells to reduce expression of target genes. However, in mammals, the artificial introduction of long dsRNAs (several hundred bp) activates the innate immune system to trigger interferon (IFN) responses. Therefore, short dsRNAs that are less likely to induce IFN responses, are typically used.

Dicer-Substrate RNAi technology. Traditional siRNA designs use duplexed, chemically-synthesised 21mers that structurally resemble endogenous siRNAs, the end product of Dicer cleavage. Dicer-substrate RNAs (DsiRNA (IDT)) are an alternative to traditional 21mer siRNAs, with an increased effectiveness of up to 100-fold compared to conventional 21mer designs, and are able to effectively target some sites that 21mers cannot6.

DsiRNAs are 27mer RNA duplexes with a novel asymmetric design that allows them to be processed by Dicer into the desired, 21mer siRNA product. DsiRNAs duplexes have a single 2-base, 3' overhang on the antisense strand and are blunt on the other end. The blunt end is further modified with DNA bases. This design provides Dicer with a single favorable binding site that helps direct the cleavage reaction.

The functional polarity introduced by this processing event favours antisense strand loading into RISC. It is thought that the increased potency of DsiRNAs is related to this linkage between Dicer processing and RISC loading7 that is, increased antisense loading will result in increased target mRNA cleavage.

Designing siRNAs

The ability of a particular siRNA to silence gene expression is predominantly determined by its sequence, and not all target sites are equal6, 9. In addition to the sequence, other considerations, such as cross-hybridization and chemical modifications, can alter the effectiveness of an siRNA 5.

Location: The localization of an siRNA within a target gene, either internal or at 5' or 3' ends, is not a major factor for silencing efficacy. However, complete knowledge of specific splice variants is important for targeting of the desired isoform(s) of the gene, as is locations of common polymorphisms8.

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, thus increasing siRNA stability10. They can also reduce siRNA activation of an innate immune response during in vivo applications9.

Additionally, modification can be used to increase cellular uptake and to prevent unwanted participation in miRNA pathways that create off-target effects9. However, chemical modification can also alter the potency of an siRNA and modified siRNAs should be empirically tested to ensure that 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 modification. That said, 5'-OH ends are rapidly phosphorylated by cellular kinases, in vitro or in vivo, so it is not necessary to phosphorylate synthetic siRNAs5.

Thermodynamic stability: The most effective siRNAs have a relatively low melting temperature (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 strand5. When options for a target sequence are limited, 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 non-complementary bases are introduced, it is important that they are on the 3'-end of the passenger strand rather than the 5'-end of the guide strand to avoid impairing the ability of the guide strand to anneal to the target.

Sequence characteristics and specificity: To maintain specificity, the guide strand should not contain sequence characteristics such as homopolymeric runs (those with 4 or more identical nucleotides) or 9-base or greater segments of G/C bases5. In addition, target accessibility eg, secondary structure, is an important factor affecting siRNA efficacy5. A moderate to low GC content (30-52 per cent) is typically a feature of functional siRNAs5.

It is important to screen candidate siRNAs for homology to other targets and exclude those with significant complementarity5. BLAST is not a good tool for finding short 5- to 8-base domains of sequence identity between a candidate siRNA and other genes; the Smith-Waterman algorithm is recommended for siRNA homology screening instead5. Programs such as SSEARCH or JALIGNER are two free options for this type of analysis.

Like targeted effects, off-target effects (OTEs) are dose dependent. Therefore, it is important to establish dose-response profiles for all siRNAs; always use the lowest concentration of siRNA that will provide sufficient target knockdown. An additional measure to identify OTE bias is to ensure that at least two, and ideally three, independent siRNAs that target different sites of a specific target RNA transcript produce the same results5.

siRNAs used in vivo show great potential as both research tools and as therapeutic agents11. For a review on the status of RNAi in therapeutics, see the recent article by Vaishnaw et al12. Before you begin RNAi studies in vivo, consider the following issues: site selection, siRNA design and chemistry, controls, route of administration, and use of a delivery vehicle11.

To find the best candidates, it is important to validate siRNA duplexes in vitro before moving to in vivo experiments.

In addition, choose more than one effective siRNA for each target to be tested, to rule out false positive results caused by off-target effects11.

RNAi is a powerful tool for studying gene silencing and its effects. Advancements to the technology, such as IDT DsiRNAs, have led to even greater improvements 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.

Jaime Sabel and Hans Packer are Scientific Writers at Integrated DNA Technologies (IDT), Coralville, IA, USA. http://eu.idtdna.com

DEFINITIONS

DsRNA - double-stranded RNA.

Dicer - an endoribonuclease that degrades long dsRNAs into small, effector molecules called siRNAs.

SiRNA- small interfering RNA.

RISC - RNA-Induced Silencing Complex. Passenger strand - the sense strand of the siRNA; degraded by Dicer during RNAi processing.

Guide strand - the antisense strand of the siRNA; incorporated into RISC during RNAi processing.

REFERENCES

1. Hannon GJ and Rossi JJ. (2004) Unlocking the potential of the human genome with RNA interference. Nature, 431(7006): 371-378.

2. Meister G and 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.

4. Whitehead KA, Langer R, and Anderson DG. (2009) Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov, 8(2): 129-138.

5. Peek AS and Behlke MA. (2007) Design of active small interfering RNAs. Curr Opin Mol Ther, 9(2): 110-118.

6. Kim DH, Behlke MA, et al. (2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol, 23(2): 222-226.

7. Rose SD, Kim DH, et al. (2005) Functional polarity is introduced by Dicer processing of short substrate RNAs. Nucleic Acids Res, 33(13): 4140-4156.

8. Amarzguioui M, Lundberg P, et al. (2006) Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc, 1(2): 508-517.

9. Behlke MA. (2008) Chemical modification of siRNAs for in vivo use. Oligonucleotides, 18(4): 305-319.

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. Behlke MA. (2006) Progress towards in vivo use of siRNAs. Mol Ther, 13(4): 644-670.

12. Vaishnaw AK, Gollob J, et al. (2010) A status report on RNAi therapeutics. Silence, 1(1): 14.





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