Stealth RNAi™ siRNA Technology

Traditional RNAi methods for gene knockdown in mammalian cells involved the use of synthetic RNA duplexes consisting of two unmodified 21mer oligonucleotides annealed together to form short/small interfering RNAs (siRNAs). Stealth RNAi™ siRNA improves upon these traditional duplexes by using proprietary chemical modifications to ensure better RNAi results. Stealth RNAi™ siRNA uses next-generation RNAi chemistry that provides higher specificity and increased stability in serum and cell culture than standard siRNA. This chemistry produces clearer results while eliminating unwanted off-target effects providing:


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Stealth RNAi™ siRNA is manufactured with the strictest quality control standards. All RNA is deprotected and desalted by normal phase (silica) chromatography and lyophilized. Trityl analysis is performed throughout the synthesis to monitor coupling efficiencies and ensure oligo quality. Each single-stranded RNA oligo is analyzed by mass spec and then annealed to deliver the specified amount of duplex. Custom Stealth RNAi™ siRNA and Stealth™ Select RNAi are delivered lyophilized, and come with 1 ml DEPC water for resuspension.

Effective Knockdown

Stealth RNAi™ siRNA provides effective knockdown to ensure silencing of the target gene. (Figure 1) compares the ability of Stealth RNAi™ siRNA with traditional siRNA for silencing. In (Figure 1), Stealth RNAi™ siRNA knockdown is highly potent just like siRNA knockdown.
Reduction Expression A549Figure 1. Reduction in expression of p53 in A549 cells by siRNA and Stealth RNAi™ siRNA delivered using Lipofectamine™ 2000.
Corresponding controls consisted of chemistry matched, scrambled sequences with a similar base-pair composition. Reduction in p53 expression is presented as fold change in expression of p53 normalized to GAPDH relative to the corresponding control measured by quantitative real-time PCR.

Higher Specificity

Off-target effects occur when the sense strand of an siRNA has homology to an untargeted gene and becomes incorporated into a RISC complex, thus directly silencing the untargeted gene. Stealth RNAi™ siRNA can eliminate sense strand off-target effects that can be problematic with traditional siRNA, even at low concentrations. These problems can arise because both the sense and antisense strand of an unmodified siRNA can enter the RNAi pathway. But Stealth RNAi™ siRNA modifications only allow the antisense strand to efficiently enter the RNAi pathway. This modification eliminates concerns about sense strand off-target effects.
RNAi-Increased SpecificityFigure 2. Stealth RNAi™ siRNA exhibits increased specificity for targets
Only the antisense strand of Stealth RNAi™ siRNA participates in RNAi knockdown, and having just one active strand limits off-target events. The sense strand of Stealth RNAi™ siRNA does not participate in RNAi activity and cannot produce off target effects.

Greater Stability

The Stealth RNAi™ siRNA modifications also increase stability when compared to traditional, unmodified siRNA. Traditional siRNAs are degraded over time in serum containing nucleases, making them undesirable for use in animals. However, Stealth RNAi™ siRNA remains stable for up to 72 hours (Figure 3) making it a better choice for projects that involve work with animal models. This flexibility can save weeks of time, avoiding the need to develop and test different molecules for animal studies and cell culture work.
RNAi More Stable SerumFigure 3.   Stealth RNAi™ siRNA is more stable in serum than standard siRNA
Stealth RNAi™ siRNA is stabilized against nuclease degradation in serum. Unmodified 21-mer dsRNA sequence (left panel) and corresponding Stealth RNAi™ siRNA sequence (right panel) at 0, 4, 8, 24, 48, and 72 hours following incubation in 10% mouse serum. Following incubation samples were separated on a Novex® 15% TBE-Urea polyacrylamide precast gel. 

Less Cellular Toxicity

Studies with standard siRNA have documented that unmodified siRNAs can induce cellular stress response pathways such as the interferon response that can result in growth inhibition and cellular toxicity. This makes it difficult to assess whether observed cellular phenotypes are due to non-specific stress responses, or to loss of function of a targeted gene. Stealth RNAi™ siRNA is a next-generation RNAi molecule that eliminates the induction of the PKR/interferon response pathway, ensuring cleaner results in RNAi experiments (Figures 4). Using Stealth RNAi™ siRNA enables potent gene knockdown without the risk of activating the cell’s stress responses that make results difficult to interpret.
RNAi Avoids InductionFigure 4. Stealth RNAi™ siRNA avoids induction of interferon response genes.
An siRNA sequence induced multiple interferon genes following delivery into A549 cells. A Stealth RNAi™ siRNA version of the same siRNA sequence did not alter expression of interferon response genes.

Summary

Concern Standard siRNA
Stealth RNAi™ siRNA
Off-target effects
Homology of the siRNA sense strand to other targets can occur, leading to degradation of mRNA other than the desired target
Only the antisense strand can participate in RNAi, avoiding unwanted off-target effects. Advanced specificity alignment of Stealth Select RNAi™ siRNA even further limits potential for off-target effects.
siRNA degradation Unmodified RNA quickly degrades in cell culture,is unlikely to have extended longevity in cell culture, and may be less effective in most in vivo applications.The Stealth RNAi™ siRNA chemical modification results in greater longevity and stability in cell culture and in serum, enhancing its use for in vivo applications.

Nonspecific toxic stress responses 		siRNA can induce nonspecific cellular stress response pathways that can lead to cell death or altered basal expression of genes, making it difficult to interpret RNAi results. Stealth RNAi™ siRNA is chemically modified to minimize the induction of nonspecific cellular stress response pathways.
Elevated false-positive and false negative results when screening Off-target and nonspecific effects can lead to false positive results. Older algorithms and bioinformatic data can lead
to increased false negatives. 		Stealth Select RNAi™ siRNA have reduced off-target and nonspecific effects and are made with the most advanced design algorithm and up-to-date bioinformatics.

Figure 5. How Stealth RNAi™ siRNA Addresses the Most Common Concerns with Unmodified 21mer siRNAs.
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