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RNAi, Methodology and its Applications

  • RNAi, Methodology and its Applications

    Hamna Shazadee, Purwa Khan, Shakra Jamil, Rahil Shahzad and Dr. Muhammad Zafar Iqabl


    RNA interference (RNAi) is a naturally occurring biological process in which RNA molecules suppress the gene expression or translation, by acting on homologous-targeted mRNA molecules in a range of organisms. This method is also known by other names including co-suppression, post-transcriptional gene silencing (PTGS), and quelling. The initial discovery of this phenomenon was in 1991, scientists who were trying to deepen the color of petunia plants. Surprisingly, when the gene for color has been introduced, they found that they had turned off the gene. Several years after the petunia experiments, the mechanism of RNA interference was revealed: this  technique was first discovered by Andrew Fire and Craig C. Mello who shared the 2006 Nobel Prize in Physiology or Medicine for their work in the nematode worm Caenorhabditis elegans, which they published in 1998.

    Two types of small RNA molecules called microRNA (miRNA) and small interfering RNA (siRNA) play key role in RNA interference. RNAs are the products of genes, and these small RNAs can bind to other RNA molecules like messenger RNA (mRNA) and either increase or decrease their activity, for example by preventing the translation of an mRNA to form a protein. RNA interference has a potential role in different mechanisms like defending cells against parasitic nucleotide sequences, viruses and transposons, to study the function of many genes associated with human disease, in research and therapy. It has become evident that RNAi has immense potential in suppression of desired genes, so this technique is now known as more precise, efficient, stable and better than antisense technology for gene silencing.

    The RNAi technology is based on the degradation of double stranded RNA molecules into short RNAs of 21 to 25 base pairs with a 2-nucleotide overhang at the 3′ end, called siRNA. This degradation is initiated by an enzyme Dicer. Each siRNA is further unwound into two single stranded RNAs (ssRNA), called the passenger strand and the guide strand. One of the strands is degraded i.e. passenger strand, while guide strand is further incorporated into an RNase complex called RNA induced silencing complex (RISC) by RISC-Loading Complex (RLC). RISC promotes this guide strand to target the complementary mRNA. As a result the targeted mRNA is not translated into the desired protein; ultimately the expression of that gene will not occur. MicroRNAs (miRNAs) are genomically encoded non-coding RNAs that can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. There are many other several key components such as RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases that have been identified in different organisms that are involved in RNAi.

    Applications of RNAi

    1.     Immune system

    RNA interference is an important part of the immune response to viruses and other foreign genetic material especially in plants which become active when plants are exposed to the different pathogens. Although animals generally express fewer variants of the dicer enzyme than plants, RNAi in some animals also play role in antiviral response. For example in Drosophila, RNA interference is important in antiviral natural immunity and act against pathogens such as Drosophila X virus. Apart from occurring naturally RNAi has also been produced artificially and is often exploited in experimental biology to study the function of genes (functional genomics) both in cell culture (in vitro) and in model organisms (in vivo). Experimentally double stranded RNA is synthesized having sequences complementary to the targeted mRNA and is introduced into cell or organism to suppress the expression of that gene. Animals like C. elegans and Drosophila, have been mostly used in functional genomics applications of RNAi as these are the common model organisms in which RNAi is most effective.

    2.    Medicine and therapeutic

    RNAi has also been used in medicines and therapy. Among the first applications in the clinical trials was the treatment of respiratory tract infection, a disease caused by the syncytial virus. Potential antiviral therapies that use RNAi to treat infection ( in mice, so far) are ; the inhibition of viral gene expression in cancerous cells, knockdown of host receptors and co-receptors for HIV,  the silencing of hepatitis A  and hepatitis B genes, silencing of influenza gene, and inhibition of measles viral replication. RNA interference has also promised to treat cancers by silencing genes in tumor cells or genes that are involved in cell division.

    3.    Biotechnology

    RNAi has also been widely used in biotechnology. A number of novel crops have been developed by using RNAi like nicotine free tobacco and nutrient fortified crops. Another important application of the RNAi is the development of genetically modified arctic apples (about to receive US approval). The apples were produced by RNAi suppression of PPO (polyphenol oxidase) gene that is responsible for browning of apple after being sliced. PPO-silenced apples are unable to convert chlorogenic acid into quinone product. Furthermore, RNAi has been used in producing genetically engineered cotton whose seeds contain reduced levels of delta-cadinene synthase, a key enzyme in gossypol production, which is a toxic compound that make cotton unsuitable for human consumption, despite of having high amount of dietary protein seeds. There are many other opportunities for the applications of RNAi in terms of crop improvement such as stress tolerance and increased nutritional level and may also be useful in inducing early flowering, delayed ripening, delayed senescence, breaking dormancy and stress-free plants etc.

    4.    Insecticides Development

    RNAi is under development as an insecticide, by using multiple approaches, including genetic engineering and topical application. In genetic engineering, transgenic crops are being developed in which dsRNA is introduced that can silence the genes in target pests. These dsRNA are synthesizes such that they only affect those insects having particular nucleotide sequence complementary to that of dsRNA, avoiding the off-target effects. While, in topical approach alternatively dsRNA is supplied without genetic manipulation, by adding them into irrigation water. The dsRNA molecules are absorbed by the plants vascular tissues and poison the insects when feed on them.

    Potential Threats

    A key area of research in the use of RNAi for clinical applications is the development of a safe delivery method for dsRNA, which has involved mainly viral vector so far, systems similar to those suggested for gene therapy, which has some safety concerns.  Any of these viral vectors can result in the activation of the body immune system if the vector is seen as a foreign invader. Furthermore, when viral vectors are used once, they cannot be effectively used in the patient again because it will be recognized by the body. Due to these safety concerns, non-viral vector methods are under development like, lipid based and polymeric vectors are also promising vectors.

    Another concern of this technology is the possibility of “off-target” effects in which a gene with a similar sequence to the targeted gene is also repressed. A computational genomics study estimated that the error rate in RNAi of off-target interactions is about 10%. In an experiment of liver disease in mice it was reported that 23 out of 49 distinct RNAi treatment protocols resulted in death.

    The role of RNAi has changed dramatically over the past two decades. Moving from transgenic plant experiments to a systematic way to silence genes for curative medicinal therapies, RNAi has been tested in a diverse range of organisms from the microscopic to plants and mammals. For our healthcare system, these therapies hold much promise. The RNAi phenomenon has not only allowed researchers to identify the genes involved in diseases, but also offers the possibility to treat complex diseases such as cancer and HIV by making the causative gene silence. However, in order for these new therapies to become fixed in our system, new policies and a revised infrastructure must be established earlier. Researchers agree some of the key challenges facing RNAi viability, stability, and specificity of the therapies and treatments. However, if such problems are addressed sooner, RNAi will bring more than our expectations in our life systems.


    1. Ding, S. W., Li, H., Lu, R., Li, F., & Li, W. X. (2004). RNA silencing: a conserved antiviral immunity of plants and animals. Virus research102(1), 109-115.
    2. Castanotto, Daniela, and John J. Rossi. “The promises and pitfalls of RNA-interference-based therapeutics.”Nature 457, no. 7228 (2009): 426-433.
    3. Chiu, Ya-Lin, and Tariq M. Rana. “RNAi in human cells: basic structural and functional features of small interfering RNA.”Molecular cell 10, no. 3 (2002): 549-561.
    4. Qiu, Shibin, Coen M. Adema, and Terran Lane. “A computational study of off-target effects of RNA interference.”Nucleic acids research 33, no. 6 (2005): 1834-1847.
    5. Dykxhoorn, Derek M., and Judy Lieberman. “The silent revolution: RNA interference as basic biology, research tool, and therapeutic.” Rev. Med. 56 (2005): 401-423.6: (5): 674



    About Rahil Shahzad

    Assistant Research Officer Agricultural Biotechnology Research Institute, AARI, Faisalabad.

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