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Overview of RNAi and Relationship to siRNAs
Since the original discovery of Fire and Mello, there have been a number of theories regarding the mechanism of gene silencing as a result of RNA interference. The most widely accepted model involves two stages: 1) an initiation step and 2) an effector step. The initiation step consists of the injection of long double-stranded RNA molecules into the cell (via injection, electroporation, lipofection, etc.). The double-stranded RNA (dsRNA) is then cleaved into short RNA duplexes (21-23 nucleotides) by the DICER enzyme (RNase III family of dsRNA-specific ribonucleases). In some cases, the dsRNA may induce a non-specific interferon response resulting in non-specific gene silencing. To prevent or minimize this non-specific response, small interfering RNAs (siRNAs) can be injected directly into the cell. In order to render the siRNAs functional, cellular kinase proteins are used to add a 5' phosphate group to the RNA duplex, producing a 19-21 base-pair siRNA duplex possessing a 2 nucleotide, 3' overhang.
During the effector step, siRNA molecules and cellular proteins combine to form the RNA Induced Silencing Complex (RISC). The RISC is believed to target and destroy mRNA transcripts by complementary base pairing with the siRNA molecule and subsequent denaturation (via ATP-dependant helicase) thus activating the RISC complex. Once the RISC is activated, the antisense strand of the siRNA directs the RISC to the target mRNA through complementary base-pairing. Although researchers now know that the cleavage of the mRNA molecule takes place approximately 12 nucleotides from the 3' end of the siRNA molecule, the exact mechanism of cleavage remains unclear.
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