1. The Mechanisms and Applications of RNAi Gwendolyn Bishop, Mary Pham, Everett Oliver, Rebecca Smith, Sabbie Sandhu, Ashley Wong University of Maryland, College ParkBCHM 465 Prof. Kahn INTRODUCTION RNA interference (RNAi) is a molecular biology mechanism where the presence of certain fragments of double-stranded RNA (dsRNA) interferes with the expression of a particular gene which shares a sequence with the dsRNA that is homologous to that gene. The revolutionary finding of RNAi resulted when plant scientists in the USA and the Netherlands tried to produce petunia plants with improved flower colors by introducing additional copies of a gene encoding a key enzyme for flower pigmentation into petunia plants. When the scientists ended up with fully or partially white flowers they discovered that both types of genes, the endogenous and the newly introduced transgenes, had been turned off. A few years later plant virologists made a similar observation. In their research they surprising observation that plants carrying only short regions of viral RNA sequences not coding for any viral protein showed enhanced tolerance or even resistance against virus infection. They concluded that viral RNA produced by transgenes can also attack incoming viruses and stop them from multiplying and spreading throughout the plant. They called this phenomenon “virus-induced gene silencing” or simply “VIGS”. These phenomena are collectively called post transcriptional gene silencing. Many laboratories around the world searched for the occurrence of this phenomenon in other organisms. Scientists A. Fire and C. Mello injected double stranded RNA into C. elegans and noticed a potent gene silencing effect. They coined the term RNAi. APPLICATIONS 1. Functional Genomics: RNA interference can be used as a tool for determining gene function. According to the Central Dogma of molecular biology, DNA is transcribed into RNA which is then translated into protein. Proteins and some RNAs are the functional components within a cell, thus the ability to selectively destroy RNA allows researchers to effectively repress gene expression. Previous methods of studying gene function have involved manipulation of DNA. A typical experiment required the generation of random mutants by exposing a group of organisms to mutagens or through the use of DNA inserts. The few that expressed a phenotypic change relevant to a given study were then selected and the genetic location of the sequence change would be determined. The advent of RNAi-based gene silencing, along with its use of genome sequence information, has dramatically changed how these tests are done. RNAi is a form of reverse genetics, meaning researchers can systematically pick genes rather than beginning with mutants and then searching for the genes affected. One major advantage of this method is that the genetic location of any given “mutation” is predetermined, which removes a large portion of previous labor. Additionally, all genes can be analyzed. If a gene is vital, a mutant might not survive long enough to be noticed, let alone studied. With RNAi, researchers can pick a chromosome and systematically knock out genes as they appear sequentially. Various methods exist for inserting dsRNA into target organisms. One method is direct injection which, though currently used for medical applications, is not the best option for high throughput research. Viruses can also inject an appropriate duplex. There are two ways of expressing dsRNA in bacterial vectors: by encoding a hairpin structure or by use of a dual promoter that expresses both the sense and the anti-sense strand. In an experiment conducted on C. elegans in 2000, dual promoters where inserted into E. coli plasmids. The worms that ate these bacteria contained the dsRNA for several days and it was carried on to their progeny that were produced within this time period. The embryos were taped as they developed and any changes relative to wild type phenotype were recorded. 2. Macular Degeneration: Macular degeneration is when the protein, vascular endothelial growth factor or VEGF, is overproduced in the eye. An excess of VEGF causes a build up of blood vessels behind the retina leading to blurred vision and possible blindness. In order to destroy the mRNA that codes for VEFG, dsRNA is injected into the whites of the eyes which then leads to reduced blood vessel formation and the shrinkage of present blood vessels. The first RNAi treatment trial for macular degeneration started in 2004, where a quarter of the participants saw a significant improvement in their vision after two months. Presently, Acuity Pharmaceuticals, Inc. is sponsoring a study with siRNA called Cand5 for the treatment of macular degeneration. They are currently in phase I where 20-80 people use the treatment to determine safety, side effects and proper dose amount. If research and trials go according to plan an RNAi treatment for macular degeneration could be available for the public as soon as 2009. Macular degeneration is one of the first diseases to test RNAi as a treatment, the reason being that the RNA can be directly injected into the diseased tissue. When the RNA has to travel through the body to get to the target site the RNA has a better chance of getting degraded or affecting the wrong gene. MECHANISM In Non-mamlian Species: A. Introduction of dsRNA triggers the RNAi Pathway. B. Dicer (cytoplasmic Nuclease) cleaves the dsRNA, thus produces siRNA(21-23bp) C. siRNA unwinds and assembles into RISC (RNA Induced Silencing Complex). D. Antisense siRNA strand then guides the RISC to complementary RNA molecules. E. RISC cleaves the mRNA. F. This leads to specific gene silencing. Mammalian Systems: Since some mammalian cells mount a potent antiviral response upon introduction of dsRNA longer then 30bp, researchers transfect cells with 21-23bp siRNA’s thereby inducing RNA in these systems without eliciting an antiviral response. FUTURE APPLICATIONS A lot of research is currently being conducted investigating the use of RNAi as a future cancer therapeutic. Results from in vitro and in vivo animal studies look promising. This method is appealing due to the specificity of RNAi in silencing target genes without affecting other genes. As more genes involved in causing cancer are being discovered and sequenced the efficiency of RNAi increases. RNAi regulates gene expression thus having the capability to inhibit expression of protein encoding genes involved in cancer. The ability of RNAi to specifically silence targeted genes makes it a potentially highly effective method of treating cancer. Research is being conducted to design specific siRNA that targets telomerase. Telomerase is an enzyme that produces telomeres which are tandem repeats of DNA (TTAGGG) located at the ends of chromosomes. Under normal cell division telomeres shorten with each round of cell division because DNA polymerase cannot incorporate nucleotide bases in the 3’  5’ direction, and thus cannot replace the 5’ RNA primer with DNA, resulting in a loss of genetic information. Consequently with each replication, telomere sequences are shortened. When telomeres reach critical shortening, DNA sensing molecules are activated and initiate an intracellular mechanism that leads to cell cycle arrest and replicative senescence. Telomerases increase telomere length, thereby enabling cancerous cells to evade senescence and allows enhanced replicative potential resulting in virtual immortality. Inhibition of telomerase activity prevents telomere extension, potentially causing replicative senescence and apoptosis of cancerous cells. Altering the telomerase will hinder cancer development and essentially make the cancerous cells susceptible to “old age”. The future use of siRNA is appealing since there are very few or no effects on normal diploid cells. However, only in vitro and animal studies have been conducted so far. Intraocular injections of RNAi targeting vascular endothelial growth factor inhibited 60% neovascularization (green fluorescence) in laser-induced rupture of retinal membranes, which mimics the growth and leakage of blood vessels behind the retina in AMD. An additional future application of RNAi includes inhibition of viral infections, specifically HIV infection, whereby proteins critical to HIV’s survival are targeted. Long strands of RNAi (approximately 500 bp in length) often illicit an interferon response in the cells. Accordingly, the “Dicer” step of RNAi can be bypass by using plasmid derived short strands of RNAi (siRNA). The siRNA’s, which are approximately 21 -25 base pairs, then destroy complementary HIV mRNA’s, thereby effectively silencing the correlated gene sequences; ultimately resulting in inhibition of HIV replication and/or its ability to attach to immune cells. Due to HIV’s ability to mutate, multiple genes can be targeted at once to ensure inhibition of HIV’s ability to make critical proteins. Namely, the HIV-1 cellular receptor CD4’s (controlled by the nef gene), the envelope associated proteins (controlled by the env gene) and the capsid proteins (controlled by the gag protein) as explained in Nature 418, 435-438 (25 July 2002). The pictorial representation to the right shows a representation of a HIV gene sequence along with a typical plasmid whereby the plasmid is used to derive the siRNAs that will target the proteins related to the gag and env gene sequences. The graph is used to show lack of antigen activity at specific gene sequences as an indication of the degree of inhibition of that gene, as noted by a study printed in Nucleic Acids Research, 2002, Vol. 30, No. 22 4830-4835 HIV gene sequences Representative bacteria plasmid by which siRNA’s are derived using PCR and purification methods. Bold horizontal bars indicate targeting by siRNA Lack of antigen activity indicates degree of gene inhibition The use of siRNA decreases the expression of telomerases. 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