Figure 1: Overview of RNA interference. The dicer enzymes produce siRNA from double-stranded RNA and mature miRNA from precursor miRNA. miRNA or siRNA is bound to an argonaute enzyme and an effector complex is formed, either a RISC (RNA-induced silencing complex) or RITS (RNA-induced transcriptional silencing) complex. RITS affects the rate of transcription by histone and DNA methylation, whereas RISC degrades mRNA to prevent it from being translated (This figure is adapted from one by Matzke MA, Matzke AJM (2004), Planting the Seeds of a New Paradigm).
Various RNAi technologies are described, along with design and methods of manufacture of siRNA reagents. These include chemical synthesis by in vitro transcription and use of plasmid or viral vectors. Other approaches to RNAi include DNA-directed RNAi (ddRNAi) that is used to produce dsRNA inside the cell, which is cleaved into siRNA by the action of Dicer, a specific type of RNAse III. MicroRNAs are derived by processing of short hairpins that can inhibit the mRNAs. Expressed small interfering RNA (siRNA) is used to express dsRNA intracellularly from DNA plasmids.
Because of its ability to silence any gene once the sequence is known, RNAi has been adopted as the research tool to discriminate gene function. After the genome of an organism is sequenced, RNAi can be designed to target every gene in the genome and target for specific phenotypes. RNAi is an important method for analyzing gene function and identifying new drug targets that uses double-stranded RNA to knock down or silence specific genes. With the advent of vector-mediated siRNA delivery methods it is now possible to make transgenic animals that can silence gene expression stably. These technologies point to the usefulness of RNAi for drug discovery.
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