siRNA / RNAi Technology? What is it?

siRNA / RNAi Technology? What is it? How does it work? What is the application? RNA interference (RNAi) represents a mechanism invented by nature to protect the genome.

Historical Aspect

siRNA was first found as part of post-transcriptional gene silencing (PTGS) in plants by David Baulcombe’s team at Sainsbury Laboratory, Norwich, England. The innovative results were first published in October 1999 in a paper entitled ‘A small RNA species for plant gene silencing post-transcription.’ Different study work has taken place in the same sequence in order to explore to their fullest extent the therapeutic potential of siRNA.

SiRNAalso commonly called short RNA or RNA silencing. A class of 20-25 nucleotide RNA double-stranded molecules of 5′ phosphate and 3′ hydroxyl (OH) group at each end that can be employed to “interfere” with the translation of proteins. This type of RNA is composed of 2′ (2 nucleotides). They bind to the specific gene sequences of mRNA and therefore act as a biotechnological tool to treat diseases such as cancer etc by promoting the degrees of specific proteins.

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Through the nucleotide sequences of their respective mRNA, they are preventing producing specific proteins. The process can also be known as “siRNA silencing or siRNA knockdown” or the RNA interference (RNAi). Si-RNA is generally regarded as coming from the long exogenous RNA strands that are taken up by the cell and subjected to further processing.

What is siRNA / RNAi Technology?

Owing to the application of the recent developments in molecular biology to the production of new pharmaceutical drugs, clinical pharmacology has undergone tremendous transformation in recent years.

The control of both the normal cell growth as well as the appearance of different alterations involving the development of diverse pathologies is essential for biochemical signalling in the cellular function. The abnormal activity of given protein results by a large number of pathologies. This would be a precondition of the treatment of these pathologies.

What is siRNA / RNAi Technology?

The RNA interference (RNAi) technology produces specific gene silencing by small, double-stranded RNA molecules. More simply, the genetic information of an individual is recorded and organized into genes in his or her DNA.

These genes are transcribed in the cell nucleus to messenger RNA (mRNA) by the genetic information of their DNA. The mRNA leaves the core of a ribosome which translates the mRNA sequence into its protein/enzyme. This translation and synthesis of proteins may be blocked if the mRNA interferes with the RNA.

RNA interference is a post-transcriptional silencing mechanism of a gene-specific type where small RNA molecules complementary to an mRNA direct degradation and thus avoid translating into proteins. RNA interference.

How does it works?

The RNA interference gene silencing is induced by small, double-stranded RNA molecules, known as siRNA (small interfering RNA) from 21 to 27 nucleotides. This siRNA suffer a series of treatment cycles inside the cell and are therefore divided into a sensory and antisense strand by their double-stranded structure.

The antisense strand is hybridized with the base-pairing specifically for the miRNA, and cellular mechanisms recognize and degrade the resulting complex. For the goal nucleotide sequence that degrades each siRNA is highly specific. This gene interference phenomenon occurs naturally throughout the organism and is involved in virus development and protection.

siRNAs can, however, also be artificially introduced into the organism to silence a particular gene. Every gene with a known sequence may serve as a target for a siRNA that includes the complementary gene sequence. As such, siRNAs are a valuable tool for the investigation of genetic function, therapeutic target validation, mechanisms for the analysis of the action of various drugs, or treatment of genetic diseases.

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RNAi is a powerful development tool with therapeutic ends mainly because of two facts: all cells have the machinery to interfere with the RNA, all genes being potential targets. Compared to various types of therapies, this technology has some advantages, including a reasonable design based on the understanding of the therapeutic objective, high specificity and reduced side effects.

In order to find molecules with therapeutic potential against different diseases, Sylentis uses this RNAi technology. Two different methods apply to the technology:

  • by means of small RNA fragments (siRNA) and
  • by means of a hairpin RNA (shRNA).

These molecules allow the particular silence meant of RNAs in messenger translation, thus inhibiting their action, which is responsible for alterations or unwanted effects in the cell.

Sylentis has developed proprietary SIRFINDER technologies that allow us, based on optimized sequence research using bioinformatics tools to reduce research time and maximize results, to design siRNAs with pharmacological potential. SIRFINDER technology A number of siRNA molecules have proven active against different therapeutic targets developed using the technique.

General Guidelines for the Design of siRNA

  1. siRNA targeted sequence is usually 21 nucleotide in length.
  2. Avoid regions within 50-100 bp of the start codon and the termination codon.
  3. Avoid intron regions.
  4. Avoid stretches of 4 or more bases such as AAAA, CCCC.
  5. Avoid regions with GC content <30% or > 60%.
  6. Avoid repeats and low complexity sequence.
  7. Avoid single nucleotide polymorphism (SNP) sites.
  8. Perform BLAST homology search to avoid off-target effects on other genes or sequences.
  9. Always design negative controls by scrambling targeted siRNA sequence.

Application of siRNA / RNAi Technology?

The seminal publication describing the use of small-scale interfering RNAs (siRNAs) in mammalian cells in 2001 revealed the use of RNA interference (RNAi) as an overall tool for gene function studies. siRNAs are the cornerstone of many research programs in less than two years’ time.

This rapid absorption was mainly caused by the easy application of siRNAs and the need for a way of reducing individual gene expression in mammalian cells so that a link could be established between gene identification and gene function. Below are some of the most important RNAi applications in mammalian systems and literature examples of these applications.

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Testing Hypotheses of Gene Function

An extensive gene database and associated phenotypes have been created for array analysis and other methods for identifying differentially expressed genes. Scientists often use expression patterns in various samples as a basis for predictions of gene function. Other mammalian gene function predictions are developed by homology research of genes known for their function in models such as Drosophila, C. And S, elegans. Cerevisiae. Cerevisiae. In many cases, it is possible to test the accuracy of these predictions with siRNAs.

Target Validation

Their almost seamless incorporation into the therapeutic development process has contributed most to the excitement of siRNAs. Drug development follows the goal identification path-> target confirmation-> therapeutic compound development-> model compound testing-> clinical trials in its simplest form. Since they are simple to use and highly specific, siRNAs provide the ultimate validation tool. The expression of a therapeutic potential goal is reduced and whether the required results of the phenotype are confident that therapeutic values should apply to an inhibitor of the same target gene.

Pathway Analysis

Pathway analysis is another important application for siRNAs. Reduce the expression of one gene has consequences for the expression and activities of genes along the same lines. For example, reducing the transcription factor levels like p53 will reduce the expression of any gene that relies on the activated transcription factor p53. In addition, gene expression regulated by p53 controlled gene products should also be affected.

The treatment of cells with a siRNA aiming at a particular gene and the subsequent monitoring of other genes using an array will allow genes associated with the target gene to be identified. Furthermore, by sequentially treating cells with siRNAs aiming at different genes and assessing which genes are affected, it is possible to disclose a specific pathway. This enables a position to be assigned for each gene in the pathway.

Gene Redundancy

In many cases, even if the gene product functions critically, the elimination of the expression of one single gene in higher eukaryotes can be tolerated. This is because more than one gene product performs many critical cell functions.

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The redundant gene product offsets a cell or animal when one gene product is eliminated. Co-transfecting siRNAs and testing for a given phenotype could be used to identify redundant genes. For example, a gene identified as important in the regulation of the cell cycle may fail to produce a defective phenotype for the cell cycle.

This siRNA can co-transfect and test with other siRNAs aimed at other genes of the cell cycle, and genes which might serve at the same point in the cell cycle could be identified for a cell phénotype. An assessment of each candidate gene alone would help to identify the most likely redundant gene, to ensure that it only causes the cell cycle defect when reduced in combination with the target gene.

Functional Screening

SIRNA Libraries targeting large gene collections will allow genes to be linked to cellular function by screening experiments. To date, the libraries have only a few large research organisations, comprising more than a few hundred siRNA.

Ambion is preparing a collection of over 1,800 siRNAs to address the well-known human kinases, recognizing the benefits of siRNA libraries. No published reports on siRNA libraries in experiments have been published, but screens were published in Drosophila and C. Examples of opportunities are elegant using dsRNA libraries.

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More than 10,000 genes in C have been used in RNAi libraries. Identification of genes regulating fat, life expectancy and control of mutations is elegant. The genes responsible for phosphorylation regulation of the down-syndrome cell-adhesion molecule were also found through a similar RNAi library for Drosophila. The keys to the tests were robust phenotypical assessments and high-quality RNAi libraries for each screening application.

siRNAs as Therapeutics: The Next Frontier

Although many investigators use siRNAs in their processes of drug development, some scientists evaluate siRNAs as therapeutic agents. When performed, siRNAs might allow virtually any gene to be targeted for therapeutic intervention.

Researchers have already shown that the RNAi pathway in mouse is active and siRNAs in various tissues are tolerated and effective. Systemically and defined tissue and target-specific responses have been injected into the synthetic siRNAs and siRNA expression vectors (both plasmid and viral).

Several publications have demonstrated that siRNAs can inhibit HIV and Hepatitis B Replication. In addition, siRNA aiming for prion-prone protein can inhibit the formation of prions in cells, thereby providing an alternative therapeutic approach to prion diseases.

Ambion will continue to develop innovative products that leverage RNAi’s power to apply fundamental, application and therapeutic research efforts as the RNAi field continues to develop and focus into animal models, therapeutic, and drug discoveries.

siRNA / RNAi Technology? What is it? – Follow us to explore more!

References

  1. S M Elbashir, J Harborth, W Lendeckel, A Yalcin, Klaus Weber, T Tuschl (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411: 494­498.
  2. Al-Khalili L, Cartee GD, Krook A (2003) RNA interference-mediated reduction in GLUT1 inhibits serum-induced glucose transport in primary human skeletal muscle cells. Biochem Biophys Res Commun. 307(1): 127­32.
  3. Chen J, Barritt GJ. (2003) Evidence that TRPC1 (transient receptor potential canonical 1) forms a Ca(2+)-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1. Biochem J. 373(Pt 2): 327­36
  4. Filleur S, Courtin A, Ait-Si-Ali S, Guglielmi J, Merle C, Harel-Bellan A, Clezardin P, Cabon F (2003) siRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth. Cancer Res. 63(14): 3919­22.
  5. De Schrijver E, Brusselmans K, Heyns W, Verhoeven G, Swinnen JV (2003) RNA interference-mediated silencing of the fatty acid synthase gene attenuates growth and induces morphological changes and apoptosis of LNCaP prostate cancer cells. Cancer Res. 63(13): 3799­804.
  6. Li K, Lin SY, Brunicardi FC, Seu P. (2003) Use of RNA interference to target cyclin E-overexpressing hepatocellular carcinoma Cancer Res. 63(13): 3593­7.
  7. Ramos-Nino ME, Scapoli L, Martinelli M, Land S, Mossman BT (2003) Microarray analysis and RNA silencing link fra-1 to cd44 and c-met expression in mesothelioma. Cancer Res. 63(13): 3539­45.
  8. Katome T, Obata T, Matsushima R, Masuyama N, Cantley LC, Gotoh Y, Kishi K, Shiota H, Ebina Y (2003) Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions. J Biol Chem. 25;278(30): 28312­23.
  9. Ashrafi K, Chang FY, Watts JL, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature. 421(6920): 268­72.
  10. Lee SS, Lee RY, Fraser AG, Kamath RS, Ahringer J, Ruvkun G (2003) A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet. 33(1): 40­8.
  11. Pothof J, Van Haaften G, Thijssen K, Kamath RS, Fraser AG, Ahringer J, Plasterk RH, Tijsterman M (2003) Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev. 15;17(4): 443­8.
  12. Muda M, Worby CA, Simonson-Leff N, Clemens JC, Dixon JE (2002) Use of double-stranded RNA-mediated interference to determine the substrates of protein tyrosine kinases and phosphatases. Biochem J. 366(Pt 1): 73­7.
  13. Caplen NJ (2003) RNAi as a gene therapy approach. Expert Opin Biol Ther. 3(4): 575­86.
  14. McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA (2002) RNA interference in adult mice. Nature 418(6893): 38­39.
  15. Song E, Lee SK, Dykxhoorn DM, Novina C, Zhang D, Crawford K, Cerny J, Sharp PA, Lieberman J, Manjunath N, Shankar P (2003) Sustained small interfering RNA-mediated human immunodeficiency virus type 1 inhibition in primary macrophages. J Virol. 77(13): 7174­81.
  16. Banerjea A, Li MJ, Bauer G, Remling L, Lee NS, Rossi J, Akkina R (2003) Inhibition of HIV-1 by lentiviral vector-transduced siRNAs in T lymphocytes differentiated in SCID-hu mice and CD34+ progenitor cell-derived macrophages. Mol Ther. 8(1): 62­71.
  17. Klein C, Bock CT, Wedemeyer H, Wustefeld T, Locarnini S, Dienes HP, Kubicka S, Manns MP, Trautwein C (2003) Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA. Gastroenterology. 125(1): 9­18.
  18. Daude N, Marella M, Chabry J (2003) Specific inhibition of pathological prion protein accumulation by small interfering RNAs. J Cell Sci. 116: 2775­9. 

Rajat Singh

My name is Rajat Singh and I have created this website to help people save their time and efforts and get the best out of it. At Bioinformatics India, we plan to curate your curiosity into words, explore different segments of how-tos, Education, News, Science, Health etc.

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