The Significance of siRNA Design, Quality, and Delivery

Overview of siRNAs and RNA Interference

RNA interference (RNAi) is a natural biological mechanism wherein short inhibitory RNA (siRNA) duplexes induce potent inhibition of gene expression. These siRNA duplexes are produced naturally when an enzyme, Dicer, cleaves long double-stranded RNA (dsRNA) introduced by viral pathogens or produced from errant transcription products. The resulting 21-23 nucleotide dsRNA fragments, termed siRNAs, then associate with an RNAse-containing complex to form the RNA-induced silencing complex (RISC). The RISC unwinds the duplex and releases the sense strand. The RISC-bound antisense strand then serves as a guide for targeting the activated complex to complementary mRNA sequences. This results in subsequent mRNA cleavage and degradation. In effect, only catalytic amounts of siRNA are required for destruction of mRNA, resulting in the knockdown or silencing of the target gene and diminished protein expression.

This elegant RNAi mechanism has been quickly adopted by the research community as a method for targeted gene expression knockdown. Gene expression silencing has become a very important strategy in functional genomics. Optimized siRNA reagent kits and protocols have now made RNAi experiments fast and convenient. More importantly, the availability of extensive siRNA libraries, high-throughput screening (HTS) platforms, and bioinformatics software has enabled comprehensive identification and investigation of gene functions in various metabolic pathways and disease processes. Therefore, RNA interference is a promising technology that is revolutionizing research in functional genomics and drug discovery.

The siRNA Experimental Workflow

A typical siRNA experiment starts from the selection of a gene target and ends in the determination of knockdown efficiency, which is interpreted with respect to the objectives of the experiment. For studies that are focused on individual gene targets, there may be a variety of decision-making steps involved, as well as process optimization requirements. For the purpose of this article, we will take a high-level look at some of the important factors to consider when designing and executing your siRNA experiment. We will also describe the ways in which Sigma has been able to overcome some of the more challenging aspects of siRNA experimentation, and to make its knowledge and expertise available to customers.

Step 1: Design of siRNA

Current studies suggest that the design of an siRNA is an extremely important, if not the most important, factor for a successful RNAi experiment. It is for this reason that Sigma-Aldrich entered into an exclusive partnership with Rosetta Inpharmatics, a wholly owned subsidiary of Merck & Co., to use their best-in-class, proprietary siRNA design algorithm to design its MISSION® line of siRNAs. MISSION® siRNAs provide RNAi researchers with cutting-edge technology for enhanced performance through improved target specificity and sensitivity in gene silencing applications.

Sigma’s Partnership with Rosetta Provides Customers Access to the Following:

• Best-in-Class siRNA Design Algorithm - MISSION® siRNA designs are based on a unique and proprietary design algorithm developed by key Rosetta scientists and bioinformaticians. This tool has been optimized and upgraded based on more than three years of extensive experimental validation and testing.
• Most Current Gene Classifications – Genes targeted by the MISSION® siRNA Druggable Genome Library are based on recent NIH sequence releases of human, rat, and mouse druggable genomes and the panels are configured to accommodate continuous feedback from world-class drug development groups.
• siRNAs of high specificity – The potential for off-target effects is reduced by minimizing homology of the MISSION® siRNA seed region (nucleotide residues 2-8 in the antisense strand) to the 3’UTR region of non-target mRNAs.
• siRNAs of high sensitivity – MISSION® siRNA sequences are designed to efficiently knockdown both high and low abundance messages.

Step 2: Quality siRNA Synthesis

Well-designed siRNAs will be less efficient if not accompanied by subsequent high quality siRNA manufacturing. In most cases, high quality is assured by implementation of extensive process controls throughout the manufacturing supply chain.

For manufacture of MISSION® siRNAs, Sigma utilizes its subsidiary, Sigma-Genosys, to produce world-class siRNA oligos. The most critical reagents in the synthesis of siRNA, the amidites, are manufactured under the highest quality specifications of Sigma Fine Chemicals. Sigma-Genosys manufactures MISSION® siRNA using patented technologies for higher quality and faster turn-around times. The authenticity of the siRNA oligos is systematically verified by mass spectrometry, while precise siRNA concentrations are determined by UV spectrophotometry. The integrity of the duplex is confirmed by gel-shift assays. Finally, protocols are standardized at our worldwide manufacturing sites to ensure timely delivery of high quality MISSION® siRNAs or custom siRNAs anywhere in the world. Sigma-Genosys has the capability to manufacture up to 3 million oligonucleotides per year for rapid completion of large projects.

Step 3: Delivery of siRNA to the Cell

Once an siRNA has been designed and synthesized, the researcher’s next challenge lies in finding a way to deliver siRNAs into the cell. For transient knockdown of gene expression, Sigma has demonstrated efficient siRNA transfection and knockdown using the N-TER Nanoparticle siRNA Transfection System. This transfection system utilizes a cell penetrating peptide that binds to siRNA non-covalently to form a nanoparticle. The nanoparticle then interacts directly with lipids on the surface of the plasma membrane, allowing diffusion across the cell membrane and delivery of the siRNA directly to the cytoplasm.

Features and Benefits of the N-TER Nanoparticle Transfection System

• Direct route - Unlike most lipid-based transfection methods, this novel delivery mechanism is thought to bypass the endosomal pathway and eliminate possible compartmentalization and degradation of siRNA.
• Rapid transfection - N-TER nanoparticles complexed with nuclei acids quickly cross the cell membranes and induce rapid knockdown of the target transcript, sometimes as early as 5 hours post-transfection.
• On target - The efficient transport of the nanoparticle minimizes the amount of siRNA needed for gene knockdown and minimizes off-target effects.
• Validated - The N-TER nanoparticles have been experimentally validated in a wide variety of cell types, including primary cells, neuronal cells, differentiated cells, and non-dividing cells. However, for particular applications the conditions may need to be optimized.

Step 4: Assay for Knockdown Efficiency

Experimentally, complete knockdown of a gene is difficult to achieve and often some residual expression is observed. The efficacy of gene silencing can be measured in a variety of ways, but it is very important to verify that the effect being measured is a result from the knockdown of the targeted gene and not due to off-target effects. Assays of the gene-knockdown effect can be at the transcriptional (mRNA) level, the translational (protein) level, or the phenotypic level. In general, most mRNA assays and protein assays are performed 24 to 72 hours post transfection. However, optimal time points may need to be assessed for particular target genes or experimental conditions.

The recommended assays listed below are not intended to be comprehensive, but provide a glimpse of the different alternatives for the researcher to explore.

1. mRNA Quantitation to Monitor Transcript
   Measuring remaining mRNA levels for the gene of interest is a direct method for monitoring knockdown efficiency. Residual mRNA may be quantitated by classical methods such as quantitative RT-PCR (qRT-PCR) and Northern blotting, or by a new bDNA hybridization assay using branched DNA (bDNA) technology (QuantiGene®).
   a. Quantitative RT-PCR – The residual mRNA is amplified by RT-PCR and then quantitated. This method has the major advantage that only very small amounts of mRNA are required (nanogram level), the probes are non-radioactive, and it can be completed in less than a day.
   b. Northern Blotting – The mRNAs are separated by size in an agarose gel, transferred (blotted) onto a membrane, incubated with fluorescent or radioactive probes, and then detected using a device consistent with the probes used. This technique has the distinct advantage of revealing the molecular weight and the molecular integrity of the transcripts.
   c. bDNA analysis Using QuantiGene® Technology – This technology measures mRNA levels directly from crude cell lysates or tissue homogenates without the need to purify or amplify the mRNA. This simplifies the process by reducing the handling time, increasing retention of the sample and eliminating bias introduced by reverse transcription and PCR amplification. Luminescence readings are normalized to an endogenous reference transcript and quantitative data processing can be automated.
2. Quantitation to Monitor Protein Production
   There are a variety of options available to assay protein production levels, from the more traditional methods such as Western blotting, ELISA, and immunofluorescence, to the recent mass spectrometric techniques such as the AQUA™ and the 18O labeling technologies.
   a. Western Blotting – This assay is analogous to Southern blotting for DNA and Northern blotting for RNA. The proteins are separated by electrophoresis, and transferred to a membrane. The protein bands are then probed with labeled antibodies. Sigma-Aldrich offers over 2,000 primary antibodies against both human and mouse proteins along with a comprehensive set of labeled anti-species secondary antibodies. Optimized protocols are widely available.
   b. AQUA™ Labeling Technology – This is a mass spectrometry-based assay for the Absolute QUAntitation of proteins. An AQUA™ peptide sequence is selected from the sequence of the target protein and then chemically synthesized with a stable isotope label, such as 13C and 15N. The target protein is enzymatically digested and a known amount of the AQUA™ peptide is added to the digest as an internal standard or calibrant. The masses of the native peptide and the AQUA™ peptide usually differ by 8-12 amu, depending on the peptide sequence used. The concentration of the target protein is then calculated based on the relative MS signals of the target peptide and the isotope-labeled AQUA™ peptide.
   c. 18O Labeling Technology – This mass spectrometry-based protein assay is somewhat analogous to the AQUA™ strategy, except that this is designed for relative protein quantitation. Proteins from two different treatment conditions are digested with trypsin and then one of the samples is labeled with 18O. The masses of the two peptides differ by 4 amu and are clearly resolved by MS. The relative ion intensities of the two peptides are quantitative indicators of their relative abundance.

Conclusions

Various factors contribute to optimizing siRNA used in RNA Interference studies. Design, Quality and Delivery represent three of the most important elements that enable accurate analysis of gene knockdown. Through our ongoing commitment to the study of gene function, Sigma has forged partnerships and made investments in this area that bring these elements together to create our best in class MISSION® siRNA Libraries. The development of these products is a result of partnerships with world-class bioinformatics leader Rosetta Inpharmatics for the latest design rules, the acquisition of key intellectual property and worldwide siRNA manufacturing capabilities for unsurpassed capacity and large-project flexibility and the continuous search for new technologies and advancements from our dedicated staff of R&D scientists that pull together the entire workflow and create unique solutions that enable the study of RNA interference.

To learn more about Sigma’s products for RNAi and to contact Sigma’s RNAi experts, please visit us online at sigma.com/rnai.