RNAi Technology

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The range of diseases and disorders it might address is unprecedented; from cancer to cardiovascular diseases, neurodegenerative disorders and even HIV. Even more exciting is the potential of RNAi in agriculture. RNAi has provided a way to control pests and diseases, introduce novel plant traits and increase crop yield. Using RNAi, scientists have developed novel crops such as nicotine-free tobacco, non-allergenic peanuts, decaffeinated coffee, and nutrient fortified maize among many others. Complete plant genome sequences has brought new dimensions in genetic modification to improve plant characteristics.

Scientists believed that they could produce any gene products by just introducing foreign genes in plants, which was not always the case 1. Plant biologists found out that introducing multiple copies of a gene that codes for purple petunia flowers led, not as expected to a deeper purple hue, but rather to plants with white or variegated flowers. In another research, gene silencing was also observed when plants were infected with RNA viruses engineered to contain fragments of the plant gene. The mechanism causing these effects was not known until American scientists Andrew Fire and Craig Mello discovered that injecting double stranded ribonucleic acids dsRNA into the worm Caenorhabditis elegans triggered the silencing of genes with sequences identical to that of the dsRNA 4.

They called the phenomenon RNA interference. Re-examining the co-suppression pathway in petunia and virus-induced gene silencing revealed that all these processes led to the accumulation of dsRNAs, hence the RNAi pathway. Fire and Mello were awarded the Nobel price for Physiology or Medicine for their discovery. In addition to its roles in regulating gene expression, RNAi is used as an immune response to infection 5 and as a natural defense mechanism against molecular parasites such as jumping genes and viral genetic elements that affect genome stability 6.

Specific types of bacteria have also been shown to trigger the RNAi pathway in plants.

RNAi Technology

Figure 1. Khan and M. Issar and R. Bashyal and R. Aggarwal Virulence of Fusarium spp. Jain, A. Manimaran, K.

Use of RNAi technology to develop a PRSV-resistant transgenic papaya | Scientific Reports

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Preview this Book. Clinical Phase I and II studies of siRNA therapies conducted between and have demonstrated potent and durable gene knockdown in the liver , with some signs of clinical improvement and without unacceptable toxicity. The nanoparticle delivery system shows the most promise yet this method presents additional challenges in the scale-up of the manufacturing process, such as the need for tightly controlled mixing processes to achieve consistent quality of the drug product.

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RNA interference has been used for applications in biotechnology and is nearing commercialization in other fields. RNAi has resulted in the invention of novel crops such as nicotine-free tobacco, decaffeinated coffee, nutrient fortified vegetation, and hypoallergenic crops.

The genetically-engineered Arctic apples received FDA approval in PPO-silenced apples are unable to convert chlorogenic acid into the standard quinone product. There are several opportunities for the applications of RNAi in crop science for its improvement such as stress tolerance and enhanced nutritional level. RNAi will prove its potential for inhibition of photorespiration to enhance the productivity of C3 plants. This knockdown technology may be useful in inducing early flowering, delayed ripening, delayed senescence, breaking dormancy, stress-free plants, overcoming self-sterility, etc.

RNAi has been used to genetically engineer plants to produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. Cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol , making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase , a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is itself important in preventing damage from plant pests.

No plant products that use RNAi-based genetic engineering have yet exited the experimental stage. Development efforts have successfully reduced the levels of allergens in tomato plants [] and fortification of plants such as tomatoes with dietary antioxidants. Another effort decreased the precursors of likely carcinogens in tobacco plants. RNAi is under development as an insecticide , employing multiple approaches, including genetic engineering and topical application. Animals exposed to RNAi at doses millions of times higher than anticipated human exposure levels show no adverse effects.

RNAi has varying effects in different species of Lepidoptera butterflies and moths. To develop resistance to RNAi, the western corn rootworm would have to change the genetic sequence of its Snf7 gene at multiple sites. Combining multiple strategies, such as engineering the protein Cry, derived from a bacterium called Bacillus thuringiensis Bt , and RNAi in one plant delay the onset of resistance. Transgenic crops have been made to express dsRNA, carefully chosen to silence crucial genes in target pests.

These dsRNAs are designed to affect only insects that express specific gene sequences. As a proof of principle , in a study showed RNAs that could kill any one of four fruit fly species while not harming the other three. The International Potato Center in Lima, Peru is looking for genes to target in the sweet potato weevil, a beetle whose larvae ravage sweet potatoes globally. Other researchers are trying to silence genes in ants, caterpillars and pollen beetles. Monsanto will likely be first to market, with a transgenic corn seed that expresses dsRNA based on gene Snf7 from the western corn rootworm , a beetle whose larvae annually cause one billion dollars in damage in the United States alone.

A paper showed that silencing Snf7 stunts larval growth, killing them within days. In the same team showed that the RNA affects very few other species. Alternatively dsRNA can be supplied without genetic engineering. One approach is to add them to irrigation water. The molecules are absorbed into the plants' vascular system and poison insects feeding on them.

Another approach involves spraying dsRNA like a conventional pesticide. This would allow faster adaptation to resistance. Such approaches would require low cost sources of dsRNAs that do not currently exist. RNAi HTS technology allows genome-wide loss-of-function screening and is broadly used in the identification of genes associated with specific phenotypes.

This technology has been hailed as a potential second genomics wave, following the first genomics wave of gene expression microarray and single nucleotide polymorphism discovery platforms. With the ability to generate a large amount of data per experiment, genome-scale RNAi screening has led to an explosion of data generation rates.

The process of RNAi was referred to as "co-suppression" and "quelling" when observed prior to the knowledge of an RNA-related mechanism. The discovery of RNAi was preceded first by observations of transcriptional inhibition by antisense RNA expressed in transgenic plants, [] and more directly by reports of unexpected outcomes in experiments performed by plant scientists in the United States and the Netherlands in the early s.

The overexpressed gene was expected to result in darker flowers, but instead caused some flowers to have less visible purple pigment, sometimes in variegated patterns, indicating that the activity of chalcone synthase had been substantially decreased or became suppressed in a context-specific manner. This would later be explained as the result of the transgene being inserted adjacent to promoters in the opposite direction in various positions throughout the genomes of some transformants, thus leading to expression of antisense transcripts and gene silencing when these promoters are active.

Another early observation of RNAi was came from a study of the fungus Neurospora crassa , [] although it was not immediately recognized as related. Further investigation of the phenomenon in plants indicated that the downregulation was due to post-transcriptional inhibition of gene expression via an increased rate of mRNA degradation. Not long after, plant virologists working on improving plant resistance to viral diseases observed a similar unexpected phenomenon.

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While it was known that plants expressing virus-specific proteins showed enhanced tolerance or resistance to viral infection, it was not expected that plants carrying only short, non-coding regions of viral RNA sequences would show similar levels of protection. Researchers believed that viral RNA produced by transgenes could also inhibit viral replication. After these initial observations in plants, laboratories searched for this phenomenon in other organisms. As a result of this work, they coined the term RNAi. This discovery represented the first identification of the causative agent for the phenomenon.

From Wikipedia, the free encyclopedia. A biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Main article: MicroRNA. Main article: Three prime untranslated region. Metabolism portal Molecular and cellular biology portal. Cellular and Molecular Life Sciences. Bibcode : Sci The Nobel Prize in Physiology or Medicine Archived from the original on 20 January Retrieved 25 January Journal of Molecular Histology.

Bibcode : Natur. Nucleic Acids Research. Molecular Cell.

The microRNA Machinery. Advances in Experimental Medicine and Biology. Amplified silencing". Frontiers in Bioscience. Zhao Y, Srivastava D April Trends in Biochemical Sciences.

RNA interference

MicroRNA biogenesis: isolation and characterization of the microprocessor complex. Methods in Molecular Biology. Trends Cell Biol. Genes Dev. Genome Res. Genetics and Molecular Biology. World Journal of Gastrointestinal Oncology. Frontiers in Cellular Neuroscience. Frontiers in Psychiatry. Biological Psychiatry.

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Molecular Cell Biology 5th ed. EMBO Reports. Current Biology. Nat Cell Biol. Cell Cycle. Cytogenetic and Genome Research.