Julian's Science Experiments
  • Famous Experiments and Inventions
  • The Scientific Method
  • Home Biochemistry Experiments Biochemistry Science Fair Projects Resources Biology Jokes Warning!
       

    Small Interfering RNA (siRNA)
    Experiments, Studies and Background Information
    For Science Labs, Lesson Plans, Class Activities & Science Fair Projects
    For High School and College Students & Teachers







    siRNA Experiments and Studies

    • Nonspecific, concentration-dependent stimulation and repression of mammalian gene expression by small interfering RNAs (siRNAs) [View Experiment]
    • RNA Interference against MDR1 but not MRP1 Reverses Drug Resistance in Human Small-Cell Lung Cancer Cells [View Experiment]
    • Detection of siRNA induced mRNA silencing by RT-qPCR: considerations for experimental design [View Experiment]
    • Genomewide view of gene silencing by small interfering RNAs [View Experiment]
    • Small interfering RNA (siRNA) knockdown of CDKN2A [View Experiment]
    • Small Interfering RNA-mediated Down-regulation of Caveolin-1 Differentially Modulates Signaling Pathways in Endothelial Cells [View Experiment]
    • Short interfering RNA (siRNA): tool or therapeutic? [View Experiment]
    • Dissertation: Delivery of nucleic acids by cell penetrating peptides: application in modulation of gene expression [View Experiment]
    • Thesis: The molecular and cell biological characterization of cell-cell junctions in mesenchymally derived cells and tissues [View Experiment]
    Small Interfering RNA (siRNA)

    Definition

    Small interfering RNA (siRNA) is a class of double-stranded RNA molecules involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene.

    Topics of Interest

    See also: Gene Silencing - RNA interference (RNAi)

    Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.

    siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants". Shortly thereafter, in 2001, synthetic siRNAs were shown to be able to induce RNAi in mammalian cells by Thomas Tuschl and colleagues in a paper published in Nature. This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development.

    siRNAs have a well-defined structure: a short (usually 21-nt) double strand of RNA (dsRNA) with 2-nt 3' overhangs on either end.

    Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important tool for gene function and drug target validation studies in the post-genomic era.

    Transfection of an exogenous siRNA can be problematic because the gene knockdown effect is only transient, particularly in rapidly dividing cells. One way of overcoming this challenge is to modify the siRNA in such a way as to allow it to be expressed by an appropriate vector, e.g., a plasmid. This is done by the introduction of a loop between the two strands, thus producing a single transcript, which can be processed into a functional siRNA. Such transcription cassettes typically use an RNA polymerase III promoter (e.g., U6 or H1), which usually directs the transcription of small nuclear RNAs (snRNAs) (U6 is involved in gene splicing; H1 is the RNase component of human RNase P). It is assumed (although not known for certain) that the resulting siRNA transcript is then processed by Dicer.

    RNA activation: It has recently been found that dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. It has been shown that dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs). It is currently not known whether RNAa is conserved in other organisms.

    Innate immunity: Introduction of too much siRNA can result in nonspecific events due to activation of innate immune responses. Most evidence to date suggests that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid inducible gene I (RIG-I) may also be involved. The induction of cytokines via toll-like receptor 7 (TLR7) has also been described. One promising method of reducing the nonspecific effects is to convert the siRNA into a microRNA. MicroRNAs occur naturally, and by harnessing this endogenous pathway it should be possible to achieve similar gene knockdown at comparatively low concentrations of resulting siRNAs. This should minimize nonspecific effects.

    Off-targeting is another challenge to the use of siRNAs as a gene knockdown tool. Here, genes with incomplete complementarity are inadvertently downregulated by the siRNA (effectively, the siRNA acts as a miRNA), leading to problems in data interpretation and potential toxicity. This, however, can be partly addressed by designing appropriate control experiments, and siRNA design algorithms are currently being developed to produce siRNAs free from off-targeting. Genome-wide expression analysis, e.g., by microarray technology, can then be used to verify this and further refine the algorithms. A 2006 paper from the laboratory of Dr. Khvorova implicates 6- or 7-basepair-long stretches from position 2 onward in the siRNA matching with 3'UTR regions in off-targeted genes

    Therapeutic applications: Given the ability to knock down essentially any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic and applied biology. There are an increasing number of large-scale RNAi screens that are designed to identify the important genes in various biological pathways. Because disease processes also depend on the activity of multiple genes, it is expected that in some situations turning off the activity of a gene with an siRNA could produce a therapeutic benefit.

    However, applying RNAi via siRNAs to living animals, especially humans, poses many challenges. Experimentally, siRNAs show different effectiveness in different cell types in a manner as yet poorly understood: some cells respond well to siRNAs and show a robust knockdown, whereas others show no such knockdown (even despite efficient transfection).

    Phase I results of the first two therapeutic RNAi trials (indicated for age-related macular degeneration, aka AMD) reported at the end of 2005 demonstrated that siRNAs are well tolerated and have suitable pharmacokinetic properties. siRNAs and related RNAi induction methods therefore stand to become an important new class of drugs in the foreseeable future.

    In 2008, a team of researchers from Texas Tech University and Harvard University announced the development of a siRNA-based treatment that may ultimately counteract the Human Immunodeficiency Virus (HIV). Human cells infected with HIV, injected into rats, have been cured by the experimental treatment. Clinical trials on humans are expected to begin by 2010.

    In 2008 a novel DNA-siRNA delivery system that could lead to more efficient and more disease-specific vaccines against infectious diseases was developed and by researchers at The University of Texas at Austin. Biomaterials based micron size particles carrying both the DNA vaccine and the siRNA to immune cells show potential to divert immune response in desirable directions

    RNA interference (RNAi) is a system within living cells that helps to control which genes are active and how active they are. Two types of small RNA molecules microRNA (miRNA) and small interfering RNA (siRNA) are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to specific other RNAs and either increase or decrease their activity, for example by preventing a messenger RNA from producing a protein. RNA interference has an important role in defending cells against parasitic genes viruses and transposons but also in directing development as well as gene expression in general.

    Transfection is the process of introducing nucleic acids into cells by non-viral methods. The term "transformation" is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells; "transduction" is often used to describe virus-mediated DNA transfer. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane, to allow the uptake of material. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Transfection can be carried out using calcium phosphate, by electroporation, or by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside. Transfection can result in unexpected morphologies and abnormalities in target cells.

    Source: Wikipedia (All text is available under the terms of the GNU Free Documentation License and Creative Commons Attribution-ShareAlike License.)

    Useful Links
    Biochemistry Resources
    Biochemistry and Cell Biology Science Fair Projects and Experiments
    General Science Fair Projects Resources
    Biology / Biochemistry Science Fair Projects Books

                  





    My Dog Kelly

    Follow Us On:
           

    Privacy Policy - Site Map - About Us - Letters to the Editor

    Comments and inquiries could be addressed to:
    webmaster@julianTrubin.com


    Last updated: June 2013
    Copyright 2003-2013 Julian Rubin