siRNA Design, Quality, and Delivery

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) into smaller fragments. 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.

RNA (dsRNA) into smaller fragments. 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.

A typical siRNA experiment starts with 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.
• Comprehensive Gene Coverage – MISSION siRNA has over 775,000 pre-designed siRNA, with multiple siRNA targeting every human, mouse, and rat gene. Finding MISSION siRNA is easy with Sigma’s “Your Favorite Gene” search engine, sigma.com/yfg.
• 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 potency – MISSION siRNA sequences are designed to efficiently knock down both high and low abundance messages.
• Guaranteed – Sigma-Aldrich guarantees that two out of three siRNA duplexes per target gene will achieve knockdown efficiencies of greater than or equal to 75%.

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, Custom Products, 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 Custom Products manufactures MISSION siRNA using patented technologies for higher quality and faster turnaround 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 Custom Products has the capability to manufacture up to 3 million oligonucleotides per year for rapid completion of large projects. For animal studies, synthesis scales of up to 100 mg and larger with purity levels up to in vivo quality are available.

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

• Flexible – with N-TER you can use your choice of cell culture media, with or without serum.
• 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.
• Efficient – The effective transport of the nanoparticle minimizes the amount of siRNA needed for gene knockdown and minimizes off-target and cytotoxic 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 a variety of methods, the most common of which is quantitative RT-PCR.
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.
   • 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.
   • Protein-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.
   • 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.

Various factors contribute to optimizing siRNA used in RNA interference studies. Design, Quality and Delivery represent three of the most important elements that allow for 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 pre-designed MISSION® siRNA and 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. The continuous search for new technologies and advancements by our dedicated staff of R&D scientists pull together the entire workflow and create unique solutions that enable the study of RNA interference.