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Chapter 4: RNAi Technology
Historically studying loss of function, phenotypes in cell culture or whole organism has been a critical aspect in determining the function of a gene. RNA interference is biological mechanism by which double-stranded RNA (dsRNA) induces gene silencing by targeting complementary mRNA. RNAi technology has revolutionized the way researchers find out the function of an unknown gene. By introducing double stranded RNA homologous to a particular mRNA, scientists can quickly and easily reduce the expression of a particular gene in nearly all organisms/cells. As compared to other conventional gene knockout strategies it allows them to quickly analyze the effect of a particular gene ( ).
In 1990, while working for a biotechnology company Napoli et al targeted to make petunias flower more purple than normal. Chalcone synthase is the key enzyme of flavonoid synthesis responsible for deep violet coloration in petunias flower. They overexpressed chimeric Chalcone synthase gene into petunia with a prediction that increasing the copy number of CHS genes would increase CHS protein levels, which would result in production of very purple flowers than wild types. Unexpectedly overexpression of chalcone synthase did not result in purple flower but leads to production of white petunia flowers. They found that the levels of CHS messenger RNA in white petunia flowers were 50 times lower than wild type purple petunia flowers. Somehow the overexpression of CHS gene (the transgene) lowered expression of both the transgene and the endogenous petunia gene. It was a mystery for almost 10 years that how overexpression of a gene can result in downregulation of its own mRNA. One theory suggested that the silencing was caused by endogenous anti-sense RNA (single-stranded RNA that is the reverse complement of mRNA). The idea was that endogenous anti-sense RNA would have base paired to the transgene mRNA and inhibited protein production ( ).
In 1998, Andrew Z. Fire and Craig C. Mello, working with C. elegans stated that trigger for gene silencing was not single stranded RNA but was double stranded RNA and double stranded RNAs can trigger silencing of complementary messenger RNA sequences. They found that although both sense and antisense single strands produced modest RNA interference, double stranded mixtures produced potent and specific interference. They further reported that these potent and specific effects were also evident in both the injected animals and their progeny. The Nobel Prize in Physiology or Medicine (2006) was awarded jointly to Andrew Z. Fire and Craig C. Mello "for their discovery of RNA interference - gene silencing by double-stranded RNA". It was initially thought that this approach would not be applicable in mammals as dsRNA molecules that are longer than 30 bp when used as siRNA in mammals resulted in the global shutdown of protein synthesis. In 2001, it was shown by Elbashir et al and Caplen and colleagues that chemically synthesized short dsRNA molecules of 21–22 nucleotides (siRNAs) could be used to silence genes in mammalian system without global shutdown of protein synthesis. Today RNAi is one of the fastest advancing fields in molecular biology research with flow of discoveries giving true meaning to the expression ‘from the workbench to the bedside’. It is being described as a powerful and promising technology for both basic research and therapeutic intervention ( ).
In many non-mammalian systems, introduction of long double- stranded RNA (dsRNA) can triggers the RNAi pathway. Dicer ( a cytoplasmic nuclease) first cleaves these long dsRNA into 21–23 bp small interfering RNAs (siRNAs), and then unwind these siRNAs and assemble into RNA-induced silencing complexes (RISCs). The antisense strand of siRNA guides RISC to complementary RNA molecules, and activated RISC cleaves targeted mRNA, leading to specific gene silencing. The strand of the siRNA that is complementary to the target mRNA sequence(s) is known as “Guide strand” while the other strand is known as sense strand or “Passenger strand”. The 5’ region of the guide strand of an siRNA, extending from nucleotides 2-7 (Hexamer) or 2-8 (heptamer) is known as “seed” sequence. In most mammalian cells, RNA interference can be induced by transfecting cells with siRNAs (typically 21 bp RNA molecules with 3’ dinucleotide overhangs) or by using DNA-based vectors to express short hairpin RNAs, (shRNAs), that are processed by Dicer into siRNA molecules. Processed siRNAs are then incorporated into a multicomponent nuclease complex known as the RNA-induced silencing complex (RISC). Activated RISC recognizes targets mRNAs leading to specific gene silencing. Catalytic core of RISC in plants and animals (with the exclusion of single-celled organisms) is AGO2. The activated RISC-siRNA complex can silence gene expression either via post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS). PTGS is being categorized in two primary mechanisms: direct sequence-specific cleavage, and translational repression and RNA degradation. When the targeted mRNA is perfectly complementary to the siRNA, it results into direct sequence specific cleavage while translational repression and RNA degradation occur when the siRNA sequence has only limited complementarity to the target in the ‘seed’ region resulting in base pairing of activated siRNA at 3’UTR region of targeted mRNA. Activated siRNAs which are complementary to promoter regions, when present within nucleus can trigger chromatin remodelling and histone modifications resulting in transcriptional gene silencing. In mammalian cells, the details of transcriptional gene silencing via siRNAs are still under investigation. Thus RNAi acts at the level of mRNA synthesis, decreasing mRNA levels and the ability of the mRNA to be translated. (Figure 1)
Figure 1: RNAi mediated gene silencing. The processing of long double stranded RNA, endogenous micro RNA (miRNA) and plasmid derived shRNA or transfection of custom synthesized synthetic siRNA can lead to generation of active functional siRNAs. Active siRNAs associate with cellular proteins to form an RNA-induced silencing complex (RISC), which contains a helicase that unwinds the duplex siRNA in an ATP-dependent reaction. In an ideal situation the antisense strand guides RISC to the target mRNA for endonucleolytic cleavage. A RISC/RNA complex with a perfect match to a target mRNA results in mRNA degradation, whereas an RNA with a partial match functions as an miRNA and can causes translational repression or degradation of targets and off- target mRNAs.
Steps of siRNA technology
1. Trypsinize and count cells. Dilute cells in antibiotic-free complete medium to achieve the appropriate plating density. Plate cells into each well of a 96-well plate. Incubate cells at 37°C with 5 % CO2 overnight.
2. Remove culture medium from the wells of the 96-well plate and add the appropriate transfection medium (optimal concentration of siRNA duplex + antibiotic free complete medium + recommended transfection reagents) to each well.
3. Incubate cells at 37°C in 5% CO2 for 24–48 hours (for mRNA analysis) or 48–96 hours (for protein analysis)
RNAi is induced in mammalian cell either by the introduction of synthetic double stranded small interfering RNAs or by plasmid and viral vector system that express double stranded short hairpin RNAs (shRNAs). These RNAs are subsequently processed to activated siRNA-RISC comlex by the cellular machinery. A vital assumption in this approach is that the knockdown of a targeted gene is specific both at mRNA and protein level. Specific gene silencing promises the potential to harness human genome data to elucidate gene function, identify drug targets and develop more specific therapeutics. ( )
As reviewed by Leung and Whittaker in 2005, a careful selection of sequences is needed to maximize gene silencing and minimize off target and nonspecific effect. An siRNA duplex (thoughts to have laser like specificity requiring near identity between the siRNA and the target mRNA) may target more than one mRNA molecules because of sequence homologies and mismatches ( ). Off-target effects were first described by Jackson and co-workers in 2003. Thus RNAi based experiments can have less sensitivity due to partial suppression of gene expression or a lack of specificity due to suppression of nonspecific targets ( ). Using genome-wide microarray profiling as a method of detection, the authors identified modest, 1.5 to 4 fold changes in the expression of dozens of genes following transfection of individual siRNA designed to target two different genes, MAPK14 and IGF1R. Transcriptional pro¬ling revealed that each of the siRNAs produced a distinct pattern of effects on transcription. 8 siRNAs duplex targeted to MAPK14 produced a distinct expression pattern, likewise each of the 16 siRNAs duplex to IGF1R produced a unique expression pattern. They found that number of off-target genes did not correlate with the extent of target silencing, and the off-target effects could not be eliminated by decreasing the concentration of siRNA. Further, no single siRNA concentration could be found that maintained full target gene silencing while reducing off-target silencing. They reasoned that the expressions of nontarget genes were suppressed due to cross-hybridization of transcripts containing regions of partial homology with the siRNA sequence. They also reported that many of the genes which lacked any substantial sequence similarity to the siRNA were also regulated. First time they found that, the off-target gene silencing was directed by the antisense strand of the siRNA whereas for the other siRNA the off-target gene silencing appeared to be directed by the sense strand, suggesting that both the sense and antisense strands (Guide strand) of siRNA duplex can contribute to transcript silencing * ( ). It was in contradiction to earlier views that the antisense strand (Guide strand) only directs RISC to complementary mRNA while the second strand (sense strand) is degraded. In 2004 (Mittal V.) has likewise said that
Similarly, in 2003 Scacheri et al investigated the specificity of siRNA–mediated gene silencing by transfecting 10 different siRNAs corresponding to a single gene (MEN1). Unexpectedly, they detected significant and divergent changes in the level of p53 and p21 after transfecting with 10 different MEN1 siRNA in parallel. They reported that the observed effect on p53 and p21 were not related to MEN1 silencing and did not define a functional relationship between MEN1 and either p53 or p21. They further found that titration of the siRNA reduced the silencing of the MEN1 target, but did not completely abolish the effect on p53 and p21 induced by some of siRNAs and these effects were also independent of lipid agent used for transfecting siRNA. They further reported that significant changes in p53/p21 were not limited to HeLa cells only and these changes in expression were also observed in CaSki, SiHa, and MCF7 cells. Together they suggested that siRNA can induce nonspecific but sequence dependent effects by acting on other unknown targets.
In 2004 Persengiev et al reported that after transfection with 200 nm luciferase siRNA under standard conditions, out of the 33,000 genes represented on an Affymetrix U133 chip, the expression of 1154 genes increased and the expression of 689 genes decreased by ≤ 2.5-fold compared with untreated cells. By RT-PCR analysis, they further confirmed expression level of twelve genes which were either increased or decreased by luciferase siRNA and reported that treatment of cells with the transfection reagent alone did not affect expression of any of the 12 genes analyzed, indicating the effect were attributable to luciferase siRNA sequence. Further, they reported that the nonspecific effects on gene expression were dependent upon siRNA concentration in a gene-specific manner. In a similar study by Tschuch, C. et al in 2008 showed that even an siRNA (distributed by several companies and widely used for RNAi experiments as negative control) directed against exogenous GFP, resulted in deregulation of 397 genes as compared to mock transfection. They reported, 48% (190 genes) were upregulated and 52% (207 genes) downregulated stating that a large number of transcripts were deregulated due to secondary indirect effects. In out of 207 downregulated transcripts, they found only 50 mRNAs with an 8 mer homology to sense and 88 to the antisensnse GFP siRNA. Thus 33.33% of downregulated transcripts did not show any homology to both sense and antisense transcripts. They also found that these off-target effects specifically increased with increasing amounts of transfected GFP siRNA in different cell lines. Thus, their studies showed that siRNA molecules that are commercially distributed and widely used as negative controls actually targeted various endogenous genes with important roles in several pathways. The off-target effects observed by Tschuch, C. et al and Persengiev et al, can not be explained by off-target regulation because the siRNAs used in their experiments lacked significant sequence similarity to any human gene.
As stated by Jackson et al in 2006,. In 2006, the work of Birmingham et al has shed more light on off targeting. They suggested that a majority of experimentally verified off targets have a 6–7 nucleotide match to the siRNA in the so-called “seed” region, thus the overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA and off-targeting is associated with the presence of one or more perfect 3’UTR matches within the Hexamer or a heptamer seed region. Using microarray technology, they generated a database of experimentally validated off-targeted genes from the expression signatures of HeLa cells transfected with one of twelve different siRNAs targeting three different genes. They reported 347 off-targeted genes after transfection of cells with 12 different siRNA. In silico methods predicted off-target typically exceeded the number identified by microarray analysis resulting in a false positive rate of over 99% at the 79% identity cutoff. In their study the number of predicted off-targets represent more than one third of number of mRNAs in the human genome. Further comparison shows only 23 of the 347 experimentally validated off-targets were identified by in silico methods (at 79% identity cut off) which represented a false negative rate of approximately 93%. Higher cutoffs produced similarly poor overlap between experimental and in silico target prediction. Finally author has concluded that “as sheer numbers of genes that contains matches with any given siRNA seed region is very large in comparison to number of actual off-targets for that siRNA, the value of the identified parameter (by itself) is limited. The identification of additional factors that have roles in off-targeting will likely lead to development of predictive algorithms that minimize off-targets and enhance siRNA design.”
For identification of additional factor which may have roles in off-targeting, I will first discuss the work of Tchurikov N.A. et al and Yi Shuang Liu et al. In 2000, Tchurikov N.A first time reported that in contrast to gene-specific silencing observed by either expression or transfection of antiparallel double stranded RNA, potent and specific gene silencing in bacteria can be induced by expression of parallel RNA that is complementary in parallel orientation to target mRNA. Further, they reported that expression of parallel RNA was found to be more effective at producing interference than an expression of antisense RNA corresponding to the same mRNA region. Their findings strongly suggested that both parallel and antiparallel RNAs are mediators of a potent and specific repression of the target gene expression. In 2000, Tchurikov N.A published an article entitled “Generation of Kruppel phenocopies by injecting into Drosophila embryos RNA complementary to mRNA in parallel orientation” in Russian language, English translation of abstract available on NCBI Pubmed is as follow