siRNA Database and Resources for RNA Interference Studies

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The discovery of reagents to inhibit RNA synthesis and translation has revolutionized the study of biological processes. These tools include small inhibitory RNA (siRNA), small hairpin RNA (shRNA) and ribozymes. As RNA interference technology has grown, so has the list of validated RNA target sequences and reagents for study. This web site includes a database of validated siRNA target sequences and links to kits and reagents that may be used for RNA interference experiments.

siRNA Overview

RNA interference refers to the inhibition of gene expression by small double-stranded RNA molecules. This phenomenon was initially demonstrated in C. elegans, in which the injection of dsRNA molecules inhibited complementary gene expression. Though the use of siRNA has become a widely used tool for down-regulating gene expression, the existence of a naturally occurring pathway in eukaryotes has been well described. The origin of endogenous siRNA (or miRNA) may be transposons, viruses, repetitive sequences and genes. The process of producing effective endogenous siRNA is regulated by three enzymes. RNA-dependent RNA polymerases convert single-stranded RNA into double-stranded RNA. Alternatively, DNA-dependent RNA polymerases produce dsRNA by transcribing inverted DNA repeats. The resulting large RNA molecules are subject to digestion by ribonuclease III (Dicer) to produce short double-stranded siRNA molecules. Argonaute proteins are then required to bind siRNA molecules to form a complex known as RISC (RNA-induced silencing complex). RISCs may then promote epigentic silencing through RNA-directed DNA methylation or by target RNA cleavage. Though protein translation may be knocked down considerably, siRNA does not normally eliminate the expression of a gene target completely.

siRNA as a Tool

The use of RNA interference for artificially manipulating gene expression was initially limited by the activation of cellular antiviral mechanisms. Exposure of cells to sequences longer than 30 nucleotides induces interferon gene expression resulting in non-specific RNA degradation and reduced protein synthesis. However, this problem was circumvented by designing 19 to 22 nucleotide siRNA sequences. Methods for siRNA delivery into cells include liposome-based addition of purified ribonucleotides to the media or transfection of plasmid vectors designed to express siRNA molecules. Plasmid vectors rely on the use of two RNA Polymerase III promoters (U6 and H1) to drive transcription of the siRNA molecule. The target sequence (19 to 29 nucleotides) is placed in a sense and antisense orientation with a small spacer group in between (short hairpin RNA or shRNA). Once transcribed, a hairpin structure is formed that can be recognized and cleaved by Dicer. Alternatively, RNA duplexes may be transcribed without hairpin structures and directly process by the RISC. Currently, there are a variety of plasmid and viral vectors that utilize similar concepts to produce siRNA or shRNA molecules.

siRNA Design

Following the discovery of siRNA, several studies attempted to identify the optimal characteristics required for siRNA design. Some of the requirements include using sequences shorter than 30 nucleotides to avoid PKR activation, sequence stability at the 5' end of the antisense strand relative to the 3' terminus and inserting a TT overhang. Based on studies like these, a number of algorithms have been developed by academic and industrial labs to predict the most effective target sequences for a given gene. Though most of these programs are not perfect, the likelihood of obtaining a predicted sequence is superior to designing sequences without consideration of the recommended features. Synthesis and testing of multiple sequences may be required. The design of siRNA experiments may contain some potential pitfalls, thus the design should be done to include appropriate controls and measurable endpoints. A negative control should consist of a non-complementary sequence with thermodynamically similar properties as the effective siRNA sequence. When transfecting a plasmid vector to introduce siRNA or shRNA, the ratio of lipid to nucleic acid should be equal and the control vector should contain a sequence that is transcribed and processed intracellularly. Validation of the siRNA effect should also be done by measuring RNA and protein expression.

siRNA libraries

The concept of down-regulating a single gene by siRNA can be exploited by high-throughput technologies to facilitate large scale genomic studies. This can be done in a microtiter-based format or by designing libraries of siRNA or shRNA molecules. Libraries may be engineered based on validated siRNA sequences or by using randomly produced sequences and a biochemical or cellular assay to identify positive elements. If multiple iterations are used, the siRNA or shRNA may be rescued, re-introduced and identified by sequencing. This strategy may be effectively used to identify drug targets or new components of signaling pathways.

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