RNA Interference (RNAi) - Group 3 Assignment 2 PDF

Group 3: Cajulao, M., Catcatan, M., Madrid, L., Mayapit, S., Ragsac, L., Salamat, D RNA interference (RNAi) A. Background RNA interference, also referred to as RNAi, is a process where cells detect a double-stranded RNA which triggers a series of biochemical processes that result in the destruction...

Group 3: Cajulao, M., Catcatan, M., Madrid, L., Mayapit, S., Ragsac, L., Salamat, D RNA interference (RNAi) A. Background RNA interference, also referred to as RNAi, is a process where cells detect a double-stranded RNA which triggers a series of biochemical processes that result in the destruction of a matched RNA sequence. Consequently, it stops the RNA-encoded protein from being produced (National Library of Medicine, 2017). The discovery of RNAi is attributed to both Dr. Andrew Fire and Dr. Craig Mello, when they observed a mutant strain of Caenorhabditis elegans exhibiting an odd twitching behavior. Fire and Mello have been researching how to regulate gene expression. They decided to explore if adding different types of RNA sequences that matched an RNA that controls a worm muscle protein would limit synthesis of that protein and cause twitching (The Nobel Prize, 2006). In their first experiment, they inserted single-stranded RNA that was complementary to the target RNA to find out if this would prevent the cell from reading the RNA. However, the worms turned out to be perfectly normal. On the other hand, in their second experiment, they created a double-stranded RNA by combining a single-stranded RNA with a complementary strand and injecting it into the worms. The double-stranded RNA caused the worms to twitch, which they concluded that the protein had been switched off or silenced. These findings were presented in 1998 and where Dr. Fire and Mello received the Nobel Prize in Physiology or Medicine in 2006 (The Explorer’s Guide to Biology, 2021). Scientists discovered that RNAi is a natural mechanism that developed in numerous species for an array of functions. For instance, as a viral protection mechanism. Many viruses have genomes composed of double-stranded RNA and they infect by injecting their genetic material. This triggers the cell's RNAi machinery, resulting in the viral RNA to be rapidly degraded thus inhibiting the spread of the infection (Agrawal et al., 2003). RNAi is also considered a refined method of genetic control. For example, MicroRNAs (miRNAs) are small RNA fragments that are found all around the genomes of living organisms which they bind to specific RNA molecules, resulting in a double-stranded RNA that inhibits protein production. This type of RNAs is involved in numerous biological functions like cell differentiation and growth, and it also has been linked to illnesses such as cancer (Agrawal et al., 2003). In the present, RNAi is used as a technique in silencing a single gene which helps researchers study how this affects the cell. Scientists can utilize small interfering RNAs (siRNAs) to bind and degrade a natural RNA in the cell. This allows researchers to find the relevance of a specific protein by observing the biological results in its absence (The Explorer’s Guide to Biology, 2021). B. Mechanism of Action (RNA interference pathway) The process of RNA interference (RNAi) shows the mechanism of cells in using the DNA sequence in their own genes to silence it (see figure 1). This is prompted by double-stranded RNA (dsRNA) which is processed by RNAse II enzymes called Dicer and Drosha. dsRNA is first processed inside the nucleus by Drosha. It will then exit the nucleus and enter the cytoplasm to bind to Dicer which cuts it into smaller fragments known as small Bio 150 Group 3: Cajulao, M., Catcatan, M., Madrid, L., Mayapit, S., Ragsac, L., Salamat, D interfering RNA (siRNAs) or microRNAs (miRNAs) containing approximately 21 base pairs of RNA strands (UMass Chan Medical School, 2022). These small fragments of RNA will be recruited by the RNA-induced silencing complex (RISC) (National Library of Medicine, n.d.). When the RISC binds to the siRNA, one strand will be removed making the other strand free to bind to the messenger RNA (mRNA) target sequences (UMass Chan Medical School, 2022). The siRNA or miRNA will be processed by Argonaute proteins by selecting the guide strand that has a 5’ end which is less thermodynamically stable. It will then further bind to the specific site of the mRNA where nucleotides need to be degraded. This will lead to RNA mediated gene silencing resulting in the disruption of protein synthesis of the target genes (Waseem, 2022). Figure 1: RNA interference pathway (Adapted from UMass Chan Medical School, 2022) C. Implications and Limitations A normal biological process called RNA interference (RNAi) controls the expression of genes. Small RNA molecules are introduced into the cell in order to target and suppress the expression of particular genes. RNAi has demonstrated considerable potential in a number of scientific and therapeutic applications, however there are several implications and limitations to be aware of. Bio 150 Group 3: Cajulao, M., Catcatan, M., Madrid, L., Mayapit, S., Ragsac, L., Salamat, D Implications of RNAi 1. Gene Regulation and Therapeutics The advancing developments and extensive research in molecular biology paved the way for shedding a new light on the pathophysiology of illnesses. This in turn led the shift from using drugs in the traditional way in which the primary bases of pharmacological therapy are anecdotal evidence of benefit and chance discoveries, to treating human diseases with drugs formulated based on empirical evidence and researches, both extensive and intensive. It is possible that the use of RNA interference in clinical medicine will close the gap created by the continued reliance of drug development on high-throughput tests of small molecules for biological activity and will enable the direct translation of molecular discoveries into therapeutics (Rao & Sockanathan, 2005). In terms of gene regulation and therapies, RNAi has important implications. It offers a formidable tool to selectively silence disease-causing genes, opening the door to potential cures for a variety of genetic diseases, viral infections, and cancer (Aagaard & Rossi, 2007). 2. Drug Development Before introducing a new drug to the public and making it available in the market, there’s a complex and long process of development before the pharmaceutical companies or developers are able to successfully launch the new cure for a certain disease. There’s a lot of steps and protocols needed to be followed before one can formulate a new drug, starting from the identifications of the problem, followed by series of research about the target, including the discovery, refinement, and clinical testing of small molecules that may inhibit the target, as well as various clinical trials in order to test the product’s effectiveness as a cure. The use of RNAi technology at various stages of drug development has the potential to completely alter the way that drugs are developed. In vitro, RNAi has been used extensively as a potent technique for target discovery and validation, and it is currently being used in vivo to validate targets using whole organisms (Samarsky et al., 2009). Example, small interfering RNA (siRNA) molecules can be engineered to target particular and almost all disease-causing genes, paving the way for the creation of custom medicines based on a patient's genetic profile (de Brito e Cunha et al., 2022). 3. Research Tool As mentioned, RNAi aids in the development of drugs as well as in gene regulation and therapies. This suggests that RNAi may serve as an effective research tool, especially in the pharmaceutical industry. RNAi is a useful research technique for examining how genes work and how particular genes are involved in different biological processes. It enables researchers to temporarily suppress gene expression and track the outcomes. For instance, RNA interference (RNAi) can be utilized to steer an endogenous biochemical pathway towards the silencing of particular target transcripts as well as induce the production of transgenic animals (Pereira & Lopes-Cendes, 2013). 4. Agricultural Applications Agriculture could undergo a revolution thanks to RNAi since it can shield crops from pests and disease. Crop protection with RNAi technology is flexible, efficient, secure, and environmentally friendly as it can be used to manage nematodes, insects, fungi, bacteria, and viruses by using Bio 150 Group 3: Cajulao, M., Catcatan, M., Madrid, L., Mayapit, S., Ragsac, L., Salamat, D alternative techniques such as the host-induced gene silencing (HIGS) as well as spray-induced gene silencing (SIGS) (Hernández-Soto & Chacón-Cerdas, 2021). Through the process of silencing specific genes in pests, RNAi can be used to formulate a new alternative for insecticides that are environmentally friendly. It is being created and used for attacking both the genes of pests and pathogens within the plants (i.e., host-induced gene silencing) and/or as topical applications (e.g., spray-induced gene silencing) in order to improve plants by altering endogenous gene expression (Mezzetti et al., 2020). With the increasing popularity of sustainable agricultural practices, this technique may be integrated as one alternative way of managing pests and insects instead of using insecticides and/or pesticides or other harmful chemicals. Limitations of RNAi Aside from RNAi, one method that can be used for gene silencing is the CRISPR method. CRISPR also known as Clustered Regularly Interspaced Short Palindromic Repeats was found to have a crucial role in terms of the microbial innate immunity in 2007 (Prabhune, 2021). Although both methods have the capability of preventing the expression of a gene and examining the phenotypic consequences by interrogating gene function, both also have their own limitations which one should be aware of in order to be able to choose the most effective method to use. So below are the list of the limitations of the RNAi method: 1. Duration of Effect RNAi often has transitory effects, which means that they last only briefly or the effects are temporary. It could be necessary to deliver RNA molecules repeatedly and continuously to maintain long-term gene silence. This is because unlike CRISPR which totally silences the gene at the DNA level (knockout), the former method only lowers the gene expression at the mRNA level (knockdown) (Prabhune, 2021). 2. Inadequate pharmacokinetics and an unsatisfactory stability Cellular enzymes quickly break down naked RNA, so alternative delivery strategies, including lipid-based nanoparticles, viral vectors, and chemical alterations, are being considered. RNases and phosphatases can damage siRNA's phosphodiester bond. The body's endonucleases and exonucleases will swiftly break down siRNA into pieces once it is consistently given into circulation, preventing intact therapeutic siRNA from accumulating in the targeted tissue (Hu et al., 2020). Bio 150 Group 3: Cajulao, M., Catcatan, M., Madrid, L., Mayapit, S., Ragsac, L., Salamat, D Bio 150 References: Aagaard, L., & Rossi, J. J. (2007). RNAi therapeutics: Principles, prospects and challenges. Advanced Drug Delivery Reviews, 59(2–3), 75–86. doi:10.1016/j.addr.2007.03.005 Agrawal, N., Dasaradhi, P., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., & Mukherjee, S. K. (2003). RNA Interference: Biology, Mechanism, and Applications. 67(4), 657–685. https://doi.org/10.1, 28/mmbr.67.4.657-685.2003 de Brito e Cunha, D., Frederico, A., Azamor, T., Melgaço, J., da Costa Neves, P., Bom, A., … Missailidis, S. (2022). Biotechnological evolution of Sirna molecules: From bench tool to the refined drug. Pharmaceuticals, 15(5), 575. doi:10.3390/ph15050575 Hernández-Soto, A., & Chacón-Cerdas, R. (2021). RNAi Crop Protection Advances. International Journal of Molecular Sciences, 22(22), 12148. doi:10.3390/ijms222212148 Hu, B., Zhong, L., Weng, Y., Peng, L., Huang, Y., Zhao, Y., & Liang, X.-J. (2020). Therapeutic sirna: State of the art. Signal Transduction and Targeted Therapy, 5(1). doi:10.1038/s41392-020-0207-x Mezzetti, B., Smagghe, G., Arpaia, S., Christiaens, O., Dietz-Pfeilstetter, A., Jones, H., … Sweet, J. (2020). RNAi: What is its position in agriculture? Journal of Pest Science, 93(4), 1125–1130. doi:10.1007/s10340-020-01238-2 National Library of Medicine. (2017). https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/ RNA Interference (RNAi). Nih.gov. Pereira, T. C., & Lopes-Cendes, I. (2013). Medical applications of RNA interference (rnai). BMC Proceedings, 7(S2). doi:10.1186/1753-6561-7-s2-k21 Prabhune, M. (2021). RNAi vs. CRISPR: Guide to Selecting the Best Gene Silencing Method [web log]. Retrieved from https://www.synthego.com/blog/rnai-vs-crispr-guide#comparison-between-crispr-and-rn-ai Rao, M., & Sockanathan, S. (2005). Molecular mechanisms of rnai: Implications for development and disease. Birth Defects Research Part C: Embryo Today: Reviews, 75(1), 28–42. doi:10.1002/bdrc.20030 Samarsky, D., Taylor, M., Kay, M., & McCaffrey, A. (2005). RNAi in drug development: Practical considerations. In A. Fire & M. Nirenberg (Authors) & K. Appasani (Ed.), RNA Interference Technology: From Basic Science to Drug Development (pp. 384-395). Cambridge: Cambridge University Press. doi:10.1017/CBO9780511546402.030 The Explorer's Guide to Biology. (2021, October 26). What is RNA interference (RNAi)? [YouTube Video]. In YouTube. https://www.youtube.com/watch?v=xDg6pu7HWz4&t=2s The Nobel Prize. (2006, October 2). The Nobel Prize in Physiology or Medicine 2006. NobelPrize.org. https://www.nobelprize.org/prizes/medicine/2006/press-release/ UMass Chan Medical School. (2022, January 7). What is RNAi - RNAi Biology | UMass Chan Medical School. Group 3: Cajulao, M., Catcatan, M., Madrid, L., Mayapit, S., Ragsac, L., Salamat, D Bio 150 https://www.umassmed.edu/rti/biology/rna/how-rnai-works/#:~:text=The%20term%20RNA%20interfere nce%20(RNAi,%2Dstranded%20RNA%20(dsRNA). Waseem, Q. U. A. (2023). RNA interference https://microbiologynotes.org/rna-interference-or-rnai/ or RNAI. Microbiology Notes.