RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from viruses and transposons.
Biochemistry of RNA interference Numerous studies have investigated the biochemical mechanisms that underpin RNAi induced gene silencing (Tabara et al., 1999; Mourrain et al., 2000; Sijen et al., 2001). These studies have revealed that RNAi suppresses gene function by promoting degradation of specific mRNA involving highly specific and complex protein–protein interactions that occur in the RNA-induced silencing complex (RISC). Depending on the thermodynamic stability of the 5′-end, both the sense and antisense regions of a given siRNA can enter the RISC complex. However, the antisense strand of the siRNA, which is complementary to the target mRNA, serves to accurately identify the target mRNA and induces sequence-specific degradation in association with other components of RISC at the relatively thermodynamically unstable 5′-end. A key component of RISC is the protein argonaute-2 that binds to a single strand of siRNA. Argonaute-2 and the 5′ strand of the siRNA mediate the recognition of the target mRNA and, with other components of RISC, induce mRNA cleavage with consecutive suppression of protein translation
siRNA (small interfering RNA) http://en.wikipedia.org/wiki/Small_interfering_RNA Small interfering RNA (siRNA), sometimes known as short interfering RNA, are a class of 20-25 nucleotide-long RNA molecules that interfere with the expression of genes. They are naturally produced as part of the RNA interference (RNAi) pathway by the enzyme Dice r. They can also be exogenously (artificially) introduced by investigators to bring about th knockdown of a particular gene. siRNA's have a well defined structure. Briefly, this is a short (usually 21-nt) double-strand of RNA (dsRNA) with 2-nt overhangs on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group. Transfection of an exogenous siRNA is problematic, since it is only transient, and the dsRNA structure cannot easily be permanently maintained. One way of overcoming these problems 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. This transcription cassette usually uses an RNA polymerase III promoter, which direct the transcription of small nuclear RNA's, such as U6 or H1. It is assumed (although not known for certain) that the resulting short hairpin RNA (shRNA) transcript is processed by Dicer. Introduction of too much siRNA can result in non-specific events due to activation of the interferon pathway. Most papers suggest that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid inducible Gene I (RIG-I may also be involved One method of reducing the non-specific effects is by turning the shRNA into a micro RNA. Micro RNA's are naturally occurring, and, as such, tolerated better by the cell. By engineering an siRNA sequence into an miRNA structure, non-specific effects can potentially be eliminated.
miRNA (micro-RNA) http://en.wikipedia.org/wiki/MiRNA A miRNA (micro-RNA) is a form of single-stranded RNA which is typically 20-25 nucleotide long. It is thought to regulate the expression of other genes. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. The DNA sequence that codes for an miRNA gene is longer than the miRNA itself. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA). In animals, the nuclear enzyme Drosha cleaves the base of the hairpin to form pre-miRNA. The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm by Exportin 5, a carrier protein. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. In plants, which lack Drosha homologues, pri- and pre-miRNA processing by Dicer probably takes place in the nucleus, and mature miRNA duplexes are exported to the cytosol by Exportin 5.
Double-stranded RNA triggers processed into siRNAs by enzyme RNAseIII family, specifically the Dicer family Processive enzyme - no larger intermediates. Dicer family proteins are ATP-dependent nucleases. These proteins contain an amino-terminal helicase domain, dual RNAseIII domains in the carboxy- terminal segment, and dsRNA-binding motifs They can also contain a PAZ domain, which is thought to be important for protein-protein interaction. Dicer homologs exist in many organisms including C. elegans , Drosphila , yeast and humans Loss of dicer: loss of silencing, processing in vitro Developmental consequence in Drosophila and C. elegans Dicer is a conserved protein
The initiation pathway may be amplified by the cell through the synthesis of a population of secondary siRNAs using the dicer-produced initiating or primary siRNAs as templates. These siRNAs are structurally distinct from dicer-produced siRNAs and appear to be produced by an RNA-dependent RNA polymerase (RdRP).
Generation of siRNA for silencing of gene expression. (A) From top to below, chemically synthesized siRNA, long dsRNA that can be cleaved by Dicer to form siRNA, and shRNA that can be cleaved by Dicer to form siRNA. (B) From top to below, sense and antisense strands are expressed by RNA polymerase III promoter (e.g., U6 promoter) separately and form a double-stranded siRNA molecule, shRNA are expressed by RNA polymerase III promoter (e.g., U6 promoter) first and then cleaved by Dicer to form mature siRNA. Chemically synthesized siRNA, shRNA, and long dsRNA have been used to generate siRNA by introducing these molecules into cells. After entry into the cytoplasm, shRNA and long dsRNA are cleaved into 21-nt long mature siRNA by a RNase III (Dicer), which is an end-recognition endonuclease). These methods generally result in temporary silencing effects. However, long dsRNA can also elicit responses of the innate immune system such as interferon (IFN) release. To obtain stable and inducible RNAi, researchers have recently developed shRNA structures driven by U6 or H1 promoters (RNase III promoters), wherein the shRNA has 2 short duplex stems: one stem connected to a loop sequence, and the other ending with 6 or more thymidines (T) as the termination signal.
RNA polymerase III (pol III) : human U6 promoters mouse U6 promoters the human H1 promoter RNA pol III was chosen to drive siRNA expression because it naturally expresses relatively large amounts of small RNAs in mammalian cells and it terminates transcription upon incorporating a string of 3–6 uridines.
http://www.ambion.com/techlib/misc/siRNA_finder.html Target prediction The secondary structure of mRNA not only influences the maturation of pre-mRNA and the translation into protein (de Smit&van Duin, 1990; Balvay et al., 1993), it also determines the efficacy of a complimentary siRNA to access its mRNA target (Holen et al., 2002; Kretschmer-Kazemi Far & Sczakiel, 2003). Notably Heale and collegues have developed a secondary structure prediction model to identify nonaccessible mRNA sites for RNAi (Heale et al., 2005). For effective gene silencing engineering of 21-nt doublestranded siRNA with a 2-nt deoxythymideine (Ts) overhang at the 3′-end has been recommended by several groups (Chiu & Rana, 2002; Elbashir et al., 2002; Paddison et al., 2002; Khvorova et al., 2003; Reynolds et al., 2004; Ui-Tei et al., 2004), because a 3′-end overhang is more efficient in guiding dsRNA to unwind. Generally synthesised siRNA should not target introns, the 5′- and 3′-end untranslated regions (UTR), and sequences within 75 bases of the start codon (ATG). Furthermore, the guanine (G)–cytosine (C) content of the designed siRNA should be between 30% and 50% and the 5′-ends of antisense and sense strand should have high and low thermodynamic stability, respectively. Investigators should avoid internal repeats and palindromes of siRNA. At certain positions in the sense strand of the 21-nt siRNA, base preferences may be considered: an adenosine (A) at positions 3 and 19; absence of G or C at position 19; and a uracil (U) at position 10; and absence of G at position 13. Indeed, thermodynamic properties of siRNA are critical in determining its stability and gene silencing efficacy (Khvorova et al., 2003). Finally, a BLASTsearch of the appropriate genome database should be performed and low-stringency sequences should be avoided to ensure that no other unrelated genes are targeted to minimize off-target effects. Many effective and specific siRNA have been published already and can be found in the public domain.
“ High-pressure injection ” was the first strategy to demonstrate successful delivery of siRNA in vivo. A large volume (1–2mL) of saline containing unmodified siRNA is injected intravenously into the tail vein of mice within very short time (in less than 7 sec), which presumably results in the siRNA molecules being forced into several organs (mainly the liver, kidney and to a lesser degree the lung; Lewis et al., 2002). Certainly, such an approach seems to be impossible in human subjects (1000 mL saline solution containing siRNA per 10 kg of weight).
Electroporation of small RNA directly into target tissues and organs has also been developed to successfully silence gene function (Kishida et al., 2004). We and others have recently instilled siRNA directly into the airways, which was very effective in mediating gene silencing or inhibiting virus replication in the lung and thus modulating disease phenotype (Bitko et al., 2005; Li et al., 2005; Bhandari et al., 2006; Yang et al., 2006).
Viral delivery has been used extensively in gene therapy to deliver DNA to target cells. Viruses evolved to specialize in gene transduction and can also be used to ferry siRNA into cells. There are 5 main classes of viruses used in the delivery of nucleotides to cells, including the retrovirus , adenovirus , lentivirus , baculovirus , and adeno-associated-virus (AAV). Retroviruses were one of the first vectors used to transduct cells with plasmids expressing hairpin-RNA constructs. Despite the relative ease of use in vitro, use of the retrovirus in vivo has safety concerns and significant limitations. Retroviruses integrate their DNA into the host.s genomic DNA, bringing with it, the risk of mutagenesis and carcinogenesis. Two pediatric patients treated with gene therapy for x-linked severe immune deficiency syndrome (x-SCID) developed leukemia following the use of retroviral vectors. An equally daunting problem is that retroviral transduction is limited to actively dividing cells, which means that the majority of mammalian cells will not receive the siRNA.
Adenoviral vectors are commonly used in gene therapy trials. Since the adenovirus does not integrate DNA into the host.s genome, the effects are short-lived, usually lost after several cell divisions. For this reason, the adenovirus is used when a short duration of action is sufficient or desirable, such as tumor-targeting therapy. The lack of genomic integration also provides a clear safety advantage to adenoviral vectors. Despite the lowered risk of insertional mutagenesis , the adenovirus is associated with significant dose-dependent liver toxicity that can severely limit therapy. Another major disadvantage of adenoviral vectors is the dependence on specific surface receptors on the target cell which are often absent, rendering transduction impossible in many cases. Several studies have reported success in delivering siRNA to cells with an adenoviral vector using local injection. There are only a few reports of success in delivering siRNA to target cells using systemic therapy, and results were mixed.
Lentiviral vectors are a promising subclass of retroviruses that lack the risk of insertional mutagenesis and are able to transducer primary and non-dividing cells. Several studies have demonstrated the use of lentiviral vectors to deliver RNAi to target cells. There is also significant research into more accurate targeting of lentiviral vectors using envelope signals .
Baculoviruses are insect viruses that can carry large quantities of genetic information. This may allow their use for combined RNAi therapy and gene therapy. Safety concerns are less prominent with these vectors since the virus is unable to replicate or express proteins in mammalian cells. Adeno-associated viruses (AAV) are another possible vector for siRNA. These viruses do not appear to be pathogenic and can transducer non-dividing cells. Despite this, their use in in vivo delivery of siRNA is limited to stereotactic brain injections.
Liposomes and nanoparticles have been heralded as an alternative to viral delivery systems. Unmodified siRNA has a half-life of less than 1 hour in human plasma and siRNA is rapidly excreted by the kidneys. Liposomes and nanoparticles can act as envelopes to protect the siRNA from metabolism and excretion, but can also carry specific molecules designed to target the siRNA to specific tissue types. Liposomes such as Lipofectamine , cationic DOTAP , neutral DOPC have been used to carry siRNA into cells. Nanoparticles such as the cationic polymer, polyethyleneimine (PEI) have also been used to successfully deliver siRNA to target cells.
Bacterial delivery using nonpathogenic bacteria has been used to silence genes in a process known as transkingdom RNA interference ( tkRNAi ). Generally, the shRNA is produced in bacteria that invade and release the RNA into eukaryotic cells (hence the term transkingdom). The bacteria can also be engineered to carry shRNA encoding DNA plasmids. The advantages of this system include safety, trivial genetic engineering, and the ability to control the vector using antibiotics .
Finally, chemical modification of siRNA has been used to improve stability and prevent degradation by serum RNAses. Importantly, these modifications must obviously not affect the RNA interference activity of the siRNA. Chemical modifications are also being sought out that could improve upon silencing activity and/or result in better targeting to specific cell types. One of the most common modifications is the use of locked nucleic acid residues (LNA). A methylene bridge connects the 4.C with the 2.O in LNA residues. This modification increases the stability of oligonucleotides in serum, without reducing the gene silencing effect. No successful in vivo studies have been performed. A second chemical modification is the replacement of phosphodiester linkages using phosphor-sulfur connections ( phosphothioates ). This modification increases the half-life of oligonucleotides in vivo. There are several other chemical modifications that have been used to try to improve delivery of siRNA to cells. SiRNA Therapeutics has successfully used a synthetic RNA derivative with several chemical modifications against a mouse model of HBV. Clearly, research into the delivery of siRNA to target cells is still in its infancy. Until these issues are resolved, the brilliance of RNAi will be moot in the clinical arena. This realization can be tempered by the groundbreaking research being done at the bench.
Intrinsic off-target effects Although RNAi is highly specific in knocking down expression of genes, there are considerable issues rising in regards to off-target effects (Fig. 5). The most common intrinsic off-target effect induced by siRNAis caused by the failure to identify similar sequences with only few nt difference in other genes that induce unspecific silencing. Saxena et al. have shown that a 21-nt siRNA with 3 to 4 mismatched nt can still efficiently silence mRNA that are partially complimentary to the active siRNA strand (Saxena et al., 2003). Of particular significance is positions 2–8 in the mature antisense siRNAstrand, which may be strongly associated with off-targeting effects despitemismatches at other positions for respective mRNA (Birmingham et al., 2006). Therefore, the stringent design of siRNA and subsequent blast to known genomic data (e.g., NCBI gene bank) may reduce the possibility of targeting other non-specific genes. The complete experimental investigation of all possible off-target effects is difficult to achieve experimentally but employing computational analysis based on the genome and transcriptome sequence data that are available in the public domain is feasible and recommended (i.e., GenBank, Refseq, EMBL and DDBJ). Another common intrinsic off-target effect provoked by siRNA is the activation of intracellular PKR and immune pathways that are linked to toll-like receptor activation (Williams, 1997; Alexopoulou et al., 2001). PKR is activated by dsRNA longer than 30 nt, which subsequently induces the production of cytokines of the IFN family. These IFNs ultimately promote inflammatory responses and alter cell metabolism, which often results in apoptosis (Kim et al., 2004). Kim and colleagues, however, demonstrated that siRNA duplexes 27 nt in length or smaller may not induce PKR activation and subsequent IFN responses (Kim et al., 2005). Therefore, in vivo titration of effective siRNA together with employing siRNA that are less or equal to 27-nt in size may greatly minimize unwanted off-target effects. Other side-effects caused by delivery methods “ High pressure injection” and electroporation can cause significant damage to the integrity of the normal tissues and organs and thus preclude the utilisation in a clinical set-up. Liposomes/cationic encapsulated siRNA may also be toxic to the host and may cause severe host immune responses. Other emerging strategies have just recently developed, which includes chemical modification of siRNA molecules, encapsulated with different molecules (such as polyamine, basic complexes, atelocollagen, polyethylenimine and virosomes). These emerging methods are still in their infancy and need to be thoroughly investigated before used in therapeutic applications.
What good is RNAi for Us A Volume Knob for gene expression RNAi applied as an experimental technique to knockout genes Functional genomic studies Determining importance of a gene in a process (SRP54) Studies to identify genomic regulators (Read between lines in the Book of Life) Use as therapeutic tool Agriculture design of disease resistance improved nutritional and handling characteristics What can we do with this ?? RNAi is a ,powerful tool that can allow us to silence any gene. Knockout analysis; Fuctional genomic studies; Identification of regulator miRNA in cell- to be able to move towards a bettrer understanding of the book of life; Therapeutic Intervention; Agriculture; Wherever else our Imagination allows
Respiratory syncytial virus (RSV) is a major cause of respiratory illness in young children. RSV causes infection of the lungs and breathing passages. In adults, it may only produce symptoms of a common cold, such as a stuffy or runny nose, sore throat, mild headache, cough, fever, and a general feeling of being ill. But RSV infections can lead to other more serious illnesses in premature babies and kids with diseases that affect the lungs, heart, or immune system. RSV is highly contagious, and can be spread through droplets containing the virus when a person coughs or sneezes. The virus can also live on surfaces such as countertops or doorknobs, and on hands and clothing. RSV can be easily spread when a person touches an object or surface contaminated with the virus. The infection can spread rapidly through schools and child-care centers. Infants often get it when older kids carry the virus home from school and pass it to them. Almost all kids are infected with RSV at least once by the time they are 2 years old. RSV infections often occur in epidemics that last from late fall through early spring. Respiratory illness caused by RSV — such as bronchiolitis or pneumonia — usually lasts about a week, but some cases may last several weeks. Doctors typically diagnose RSV by taking a medical history and doing a physical exam. Generally, in healthy kids, it's not necessary to distinguish RSV from a common cold. But in cases where a child has other health conditions, a doctor might want to make a specific diagnosis. RSV is typically identified in nasal secretions, which can be collected either with a cotton swab or by suction through a bulb syringe. Human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus of the family Paramyxoviridae , which includes common respiratory viruses such as those causing measles and mumps . RSV is a member of the paramyxovirus subfamily Pneumovirinae . RSV causes respiratory tract infections in patients of all ages. It is the major cause of lower respiratory tract infection during infancy and childhood. In temperate climates there is an annual epidemic during the winter months. In tropical climates, infection is most common during the rainy season. In the United States, 60% of infants are infected during their first RSV season  , and nearly all children will have been infected with the virus by 2-3 years of age  . Natural infection with RSV does not induce protective immunity, and thus people can be infected multiple times. Sometimes an infant can become symptomatically infected more than once even within a single RSV season. More recently, severe RSV infections have increasingly been found among elderly patients as well.
Macular Degeneration or Macular Disease is an eye disease of the retina that is associated in which the central portion of the retina becomes damaged. Central vision is the sharpest or clearest area of your vision and is required for activities such as reading, driving and anything that is visually demanding, whether far or near. Macular Disease is quite common and affects more people in the United States than either cataracts or glaucoma. In fact, Macular Disease is the most frequent cause of blindness for patients aged 55 and above in the United States and is estimated to affect over 10 million Americans ( http://www.macular.org/disease.html ) in some fashion. As the name implies, Macular Disease affects the Macula. The Macula is the area of the retina that allows us to see fine detail and is responsible for central or “straight ahead” vision as well as the ability to see the detail of faces, reading material, colors and precise vision required for driving a car. Age Related Macular Degeneration (ARMD) is the most common type of macular degeneration. Your chances of developing Age Related Macular Degeneration (ARMD) are directly related to you age. The older you are, the greater the chance that you will be affected by Macular Degeneration. Age Related Macular Degeneration (ARMD) is a degenerative condition of the macula that results from hardening of the very fine arteries of the retina that carry oxygen and nutrients to the retina. Depriving the macula of oxygen and nutrition cause a gradual and progressive loss of function. The visual effects of macular degeneration can be relatively minimal with a mild “dimming” or “distortion” of your central vision, or very profound resulting in a complete loss of your central vision. However, macular degeneration DOES NOT cause total blindness. Since the effect of Macular Degeneration is limited to the central retina, its effects are limited to central vision without causing any disturbance of your affecting peripheral vision.
RNA interference has been used for applications in biotechnology , particularly in the engineering of food plants that produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol , making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase , a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pests.  Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.  Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants  and decreased the precursors of likely carcinogens in tobacco plants.  Other plant traits that have been engineered in the laboratory include the production of non- narcotic natural products by the opium poppy ,  resistance to common plant viruses,  and fortification of plants such as tomatoes with dietary antioxidants .  Previous commercial products, including the Flavr Savr tomato and two cultivars of ringspot -resistant papaya , were originally developed using antisense technology but likely exploited the RNAi pathway.
Isfahan University of Medical Science, School of Pharmacy Department of Clinical BiochemistryJune 25, 2012 1 Total slides : 51
O u t lin e s Introduction RNA silencing Definition of RNA interference Discovery of RNAi Mechanism of RNA interference Generation of small interfering RNA Small interfering RNA delivery methods Applications of RNA interference Therapeutic applications Other applications Conclusion
In t r o d u c t io n RN A i (R N A In t e r f e r e n c e )
RNA silencingSeveral terms are used to described RNA silencing; usually there are three phenotypically different but mechanistically similar phenomena:3. Cosuppression or post-trascriptional gene silencing (PTGS) in plants5. Quelling in fungi7. RNA interference in animal kingdom
Quelling:The silencing in fungal system Quelling came to light during attempts to boost the production of an orange pigment made by the gene al1 of the fungus Neurospora crassa N. crassa (al1+) transformed with plasmid+al1 few transformants show albino phenotype al1-quelled strain had similar level of unspliced al1 mRNA to wilde-type. Native al1 mRNA was highly reduced indicating that quelling and NOT the rate of transcription affected the level of mature mRNA in a homology- dependent manner
RNAi:Silencing in Cenorhabditis elegans dsRNA administrated to worms can permeate and affect the entire body causing a systemic RNA-interference RNAi studies represents a means of identifying partial or complete loss-of-function phenotypes, possibly leading to the identification of gene function.
Definition RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes. RNAi targets include RNA from viruses and transposons.
Need for interference Defense Mechanism Defense against Infection by viruses, etc As a defense mechanism to protect against transposons and other insertional elements Genome Wide Regulation RNAi plays a role in regulating development and genome maintenance. 30% of human genome regulated
PTGS in plants:The discovery of Jorgensen and Napoli in1990 They were trying to make petunias more purple Overexpression of petunia gene Entered homologous RNA Expected:more pigments Observed:white sectorsCosuppression:• Loss of mRNAs of both endo-and transgene
Cenorhabditis elegansRNAi can be induced in C. elegans in three simple ways: Injection of dsRNA into the worm gonads Soaking the worms in dsRNA solution Feeding the worms engineered bacteria producing dsRNA
DiscoveryInject worms with dsRNAcorresponding to a gene(important for muscle function)involved in wiggling (unc-22)
DiscoveryInject worms with dsRNAcorresponding to a gene(important for muscle function)involved in wiggling (unc-22)Conclusion: dsRNA triggers potent and specific gene silencing
RNAi vs AntisenseEffects of mex-3 RNA interference on levels of the endogenous mRNA.• Negative control showing lack of staining in the absence of the hybridization probe.• Embryo from uninjected parent showing normal pattern of endogenous mex-3 RNA (purple staining).• Embryo from parent injected with purified mex-3 antisense RNA.• Embryo from a parent injected with dsRNA corresponding to mex-3. Injected antisense or dsRNA into C. elegans dsRNA was more effective than ssRNA (antisense) Effective even in tiny amounts Inactivation was due to degradation of target mRNA
What is the secret of this little worms success?Simple organismSimilarity to other complicated animals It has neurons,muscles, gut, …Simple life cycle Rapid growth Produce about 300 progeny in 6 days Eat bacteriaSuitable for genetic investigation
What is the secret of this little wormssuccess?
Nobel prize winners in the C. elegans field Sidney Brenner John Sulston Robert Horvitz Andrew FireCraig Mello
Happy ending These results were thoroughly reproducible RNAi was found to work in many species RNAi became a powerful method that is changing the face of biology MicroRNAs were discovered (the next topic) Andy and Craig win the 2006 Nobel Prize in Physiology or Medicine!
RNAi was found to work in many diverse species Fungi Trypanosomes Insects ZebrafishMice
M e c h a n is m o f R N Ai RN A i (R N A In t e r f e r e n c e )
RNAi Overview During RNAi Double-stranded RNAs cut into short double-stranded RNAs, s(small) i(interfering) RNAs, by an enzyme called Dicer. These then base pair to an mRNA through a dsRNA-enzyme complex. This will either lead to degradation of the mRNA strand Highly specific process Very potent activity So far only been seen in eukaryotes Evidence 30% of genome is regulated by RNAi
The Players In Interference RNA siRNA: dsRNA 21-22 nt. miRNA: ssRNA 19-25nt. Encoded by non protein coding genome RISC: RNA induced Silencing Complex, that cleaves mRNA Enzymes Dicer : produces 20-21 nt cleavages that initiate RNAi Drosha : cleaves base hairpin in to form pre miRNA; which is later processed by Dicer
siRNAs Small interfering RNAs that have an integral role in the phenomenon of RNA interference (RNAi), a form of post- transcriptional gene silencing RNAi: 21-25 nt fragments, which bind to the complementary portion of the target mRNA and tag it for degradation A single base pair difference between the siRNA template and the target mRNA is enough to block the process. Each strand of siRNA has: a. 5’-phosphate termini b. 3’-hydroxyl termini c. 2/3-nucleotide 3’ overhangs
miRNA Originate from capped & polyadenylated full length precursors (pri-miRNA) Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt (miRNA)
Difference between miRNA and siRNA Function of both species is regulation of gene expression. Difference is in where they originate. siRNA originates with dsRNA. siRNA is most commonly a response to foreign RNA (usually viral) and is often 100% complementary to the target. miRNA originates with ssRNA that forms a hairpin secondary structure. miRNA regulates post-transcriptional gene expression and is often not 100% complementary to the target. And also miRNA help to regulate gene expression, particularly during induction of heterochromatin formation serves to downregulate genes pre- transcriptionally (RNA induced transcriptional silencing or RITS) RITS
Dicer RNase III-like dsRNA-specific ribonuclease • Enzyme involved in the initiation of RNAi. • It is able to digest dsRNA into uniformly sized small RNAs (siRNA) Dicer family proteins are ATP- dependent nucleases. Rnase III enzyme acts as a dimer Loss of dicer→loss of silencing processing in vitro Dicer homologs exist in many organisms including C.elegans, Drosphila, yeast and humans (Dicer is a conserved protein)
Dicer’s domainsDicer is a ribonuclease (Rnase III family) with 4 distinct domains: domains 1 4 2 2 31. Amino-terminal helicase domain2. Dual Rnase III motifs in the carboxy terminal segment3. dsRNA binding domain4. PAZ domain (110-130 amino-acid domain present in protein like Argo, Piwi..);it is thought to be important for protein-protein interaction
RISC RISC is a large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA Unwinding of double- stranded siRNA by ATP independent helicase. The active components of an RISC are endonucleases called argonaute proteins which cleave the target mRNA strand.
Mechanism of RNA interference • dsRNA are chopped into short interfering RNAs (siRNA) by Dicer. siRNA Dicer 2. The siRNA-Dicer complex recruits additional components to form an RNA-Induced Silencing Complex (RISC). The siRNA unwinds. RISC 3. The unwound siRNA base pairs with complementary mRNA, thus guiding the RNAi machinery to the target mRNA. 4. The target mRNA is effectively cleaved and subsequently degraded – resulting in gene silencing. silencing
Exogenous dsRNA is detected and bound by aneffector protein, known as RDE-4 in C. elegans andR2D2 in Drosophila, that stimulate dicer activity.Thisprotein only binds long dsRNAs.These RNA-binding proteins then facilitate transfer ofcleaved siRNA to the RISC complex.
Summary of Players Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa) Drosha and Dicer are RNase III enzymes Pasha is a dsRNA binding protein Exportin 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires Ran and GTP Argonautes are RNase H enzymes
G e n e r a t io n o f s iR N A RN A i (R N A In t e r f e r e n c e )
siRNA design 21-23nt 2-nt 3 overhangs ( UU overhangs ) G/C content: 30-50%. No basepair mismatch Synthesised siRNA should not target introns, the 5′and 3′-end untranslated regions (UTR), and sequences within 75 bases of the start codon (ATG). BLAST : eliminate any target sequences with significant homology to other coding sequences.
s iR N A d e l iv e r y method s RN A i (R N A In t e r f e r e n c e )
Effective methods for the delivery of small RNA to allow asufficient silencing effect in the target organ(s) and/or cells areyet to be developed.In particular, toxicity and side effects of RNAi must be wellcharacterized and limited.Therefore, careful design and selection of target sequence andquantification of the effect on the expression of target protein andmRNA are essential for success of gene interfering approaches.
High-pressure injection “High-pressure injection” was the first strategy to demonstrate injection successful delivery of siRNA in vivo. A large volume (1–2mL) of saline containing unmodified siRNA is injected intravenously into the tail vein of mice within very short time (in less than 7 sec), which presumably results in the siRNA molecules being forced into several organs mainly the liver, kidney and to a lesser degree the lung. Certainly, such an approach seems to be impossible in human subjects (1000 mL saline solution containing siRNA per 10 kg of weight).
Electroporation• Electroporation of small RNA directly into target tissues and organs has also been developed to successfully silence gene function.
Problem Delivery of siRNA to tissue is a problem both because: The material must reach the target organ And must also enter the cytoplasm of target cells. RNA cannot penetrate cellular membranes, so systemic delivery of siRNA is unlikely to be successful. RNA is quickly degraded by RNAse activity in serum and even siRNA chemically modified to be more stable has a half-life of only a few hours at most.
Solution For these reasons, other mechanisms to deliver siRNA to target cells has been devised. These methods include: Viral delivery The use of liposomes or nanoparticles Bacterial delivery Chemical modification of siRNA to improve stability
Viral delivery Viral delivery has been used extensively in gene therapy to deliver DNA to target cells. There are 5 main classes of viruses used in the delivery of nucleotides to cells: Retrovirus Adenovirus Lentivirus Baculovirus Adeno-associated-virus (AAV).
Retroviruses Retroviruses were one of the first vectors used to transduct cells with plasmids expressing hairpin-RNA constructs. Despite the relative ease of use in vitro, use of the retrovirus in vivo has safety concerns and significant limitations. Retroviruses integrate their DNA into the host.s genomic DNA, bringing with it, the risk of mutagenesis and carcinogenesis. carcinogenesis Another problem is that retroviral transduction is limited to actively dividing cells, which means that the majority of mammalian cells will not receive the siRNA.
Adenovirus Adenoviral vectors are commonly used in gene therapy trials. Since the adenovirus does not integrate DNA into the host.s genome, the effects are short-lived, usually lost after several cell divisions. For this reason, the adenovirus is used when a short duration of action is sufficient or desirable, such as tumor-targeting therapy. Despite the lowered risk of insertional mutagenesis, the adenovirus is associated with significant dose-dependent liver toxicity that can severely limit therapy. Another major disadvantage of adenoviral vectors is the dependence on specific surface receptors on the target cell which are often absent, rendering transduction impossible in many cases.
Lentivirus Lentiviral vectors are a subclass of retroviruses that lack the risk of insertional mutagenesis and are able to transducer primary and non-dividing cells. Several studies have demonstrated the use of lentiviral vectors to deliver RNAi to target cells.
Baculoviruses Baculoviruses are insect viruses that can carry large quantities of genetic information. This may allow their use for combined RNAi therapy and gene therapy. Safety concerns are less prominent with these vectors since the virus is unable to replicate or express proteins in mammalian cells.
Adeno-associated viruses (AAV) Adeno-associated viruses (AAV) are another possible vector for siRNA. These viruses do not appear to be pathogenic and can transducer non-dividing cells.
Liposomes and nanoparticles Liposomes and nanoparticles have been known as an alternative to viral delivery systems. Unmodified siRNA has a half-life of less than 1 hour in human plasma and siRNA is rapidly excreted by the kidneys. Liposomes and nanoparticles can act as envelopes to protect the siRNA from metabolism and excretion, but can also carry specific molecules designed to target the siRNA to specific tissue types. Liposomes such as Lipofectamine, cationic DOTAP, neutral DOPC have been used to carry siRNA into cells. Nanoparticles such as the cationic polymer, polyethyleneimine (PEI) have also been used to successfully deliver siRNA to target cells.
Bacterial delivery Bacterial delivery using nonpathogenic bacteria has been used to silence genes in a process known as transkingdom RNA interference (tkRNAi). Generally, the shRNA is produced in bacteria that invade and release the RNA into eukaryotic cells (hence the term transkingdom). The bacteria can also be engineered to carry shRNA encoding DNA plasmids. The advantages of this system include: Safety Ability to control the vector using antibiotics
Chemical modification Finally, chemical modification of siRNA has been used to improve stability and prevent degradation by serum RNAase. Importantly, these modifications must obviously not affect the RNA interference activity of the siRNA. One of the most common modifications is the use of locked nucleic acid residues (LNA). A methylene bridge connects the 4.C with the 2.O in LNA residues. This modification increases the stability of oligonucleotides in serum, without reducing the gene silencing effect.
Side effects of gene silencing by smallRNA molecules Unspecific silencing Caused by the failure to identify similar sequences with only few nt difference in other genes. Activation of intracellular PKR and immune pathways that are linked to toll-like receptor activation. PKR ( protein kinase R) is activated by dsRNA longer than 30 nt, which subsequently induces the production of cytokines of the IFN family. These IFNs ultimately promote inflammatory responses.
Side effects of gene silencing… High pressure injection” and electroporation can cause significant injection damage to the integrity of the normal tissues and organs and thus preclude the utilisation in a clinical set-up. Liposomes/cationic encapsulated siRNA may also be toxic to the host and may cause severe host immune responses. Other emerging strategies includes chemical modification of siRNA molecules and encapsulated with different molecules are still in their infancy and need to be thoroughly investigated before used in therapeutic applications.
Ap p l ic a t io n s o f R N Ai RN A i (R N A In t e r f e r e n c e )
Hematology (blood) Hematologic disorders result from Loss of gene function Mutant gene function Absent gene function RNAi May be used to create models of disease processes Could help to develop pharmacologic and genetic therapeutic targets
Oncology (cancer)Targeting of oncogenes Dominant mutant oncogenes, amplified oncogenes, viral oncogenesDefine role of signaling molecules in tumor-creationImprovement efficacy of chemotherapy andradiotherapyTumor regression through creation of potentially newmode of chemotherapy
Stem cell biology Mouse research Knock out tumor-suppression gene in mouse embryonic stem cell Observe tumor phenotype Positive correlation between extent of Trp 53 (suppression gene) inhibition and severity of disease
Infectious Diseases Virus targeting RNAi – inhibit cellular and viral factors of disease RNA transcriptase is RNAi target Inhibition of replicationMain goal Render cells resistant to infectious organisms
Hepatitis C Infects ~200 million people worldwide Often fatal 2002, Anton McCaffrey and Mark Kay at Stanford University Injected "naked" RNA strands into the tail veins of mice RNAi treatment controlled the virus in mice
Silencing genes in HIVAIM: Silence the main structural protein in the virus, p24, and the human protein CD4. Hit the virus where it counts by eliminating a protein it needs to reproduce or cause infections.
Respiratory infections RSV, infects almost every child by the age of two 2005, Sailen Barik University of South Alabama Short strands of "naked" RNA Controlled the virus in mice Clinical trials are ongoing
Macular degeneration Macular degeneration is the leading cause of adult blindness Excess VEGF which leads to sprouting of excess blood vessels behind the retina & obscuring vision. The new RNAi drugs shut down genes that produce VEGF. The drug can be injected directly into the eye First clinical trial: 24 patients, launched in 2004. Two months after being injected with the drug, 6 of the patients had significantly clearer vision Other patients vision had at least stabilized More extensive trials are ongoing
Huntington’s diseaseIdeal candidate for RNAi therapyDisease caused by protein, thataffects more than 30,000 peoplein the U.S. alone.We would want to shut down theexpression of the gene coding forthe abberant protein2004, Beverly Davidson andcolleagues at the University ofIowaDavidson treated mice withHuntingtons
Other uses of RNAi Studying cell division Testing Hypotheses of Gene Function Target Validation Pathway Analysis • Gene Redundancy Functional Screening
Studying cell division using RNAi Genes involved in cell division identified by using RNAi RNA interference (RNAi) used to assign functions of genes involved in C. elegans cell division 133 genes identified. Only 11 previously identified
Testing Hypotheses of Gene Function Array analysis and other methods for identifying differentially expressed genes have created an enormous database of genes and associated phenotypes. In many cases, scientists make predictions about gene function based on expression patterns in different samples. Other predictions of mammalian gene function are developed using homology searches with genes whose functions are known in model organisms like Drosophila, C. elegans, and S. cerevisiae. In many cases, testing the accuracy of these predictions can be accomplished using siRNAs.
Testing Hypotheses of Gene Function:……… still workingAl-Khalili et al treated myotubes with serum and showed that increased glucose uptake correlated with increased cell-surface content of glucose transporter (GLUT1). To confirm that glucose transport depends on GLUT1 expression, cells were treated with GLUT1 siRNA and were shown to have reduced levels of serum- stimulated glucose transport.Chen and Barritt used siRNAs to study the transient receptor potential canonical 1 (TRPC1) gene. The TRPC1 gene was thought to encode a non-selective cation channel activated by depletion of cellular storage and/or an intracellular messenger. When liver cells were treated with the TRPC1 siRNA, they exhibited increased cell volume and decreased inflow of Ca2+, Mn2+.
Target Validation In its simplest form, drug development follows the path of target identification → target validation → therapeutic compound development → compound testing in model systems → clinical trials. siRNA are easy to use and highly specific, they provide the ultimate tool for validation studies. Reducing the expression of a potential therapeutic target and determining if the desired phenotype results provides confidence that an inhibitor of the same target gene should have therapeutic value.
Target Validation:… an interesting example Filleur et al showed that the antiangiogenic molecule thrombospondin-1 (TSP-1) could reduce vascularization and delay tumor onset. Over time, tumor cells producing active TSP1 began to form exponentially growing tumors. These tumors were composed of cells secreting unusually high amounts of the angiogenic stimulator, vascular endothelial growth factor (VEGF), which were sufficient to overcome the inhibitory TSP1. Treating tumor cells with a combination of TSP1 and a VEGF- specific siRNA caused a striking reduction in cell proliferation. This result suggested that using a combination of TSP1 and an anti-VEGF compound would slow or eliminate tumor growth
Pathway Analysis Reducing the expression of a single gene has implications on the expression and activities of genes that are in the same pathway(s). Treating cells with an siRNA targeting a given gene and then monitoring the expression of other genes using a microarray will make it possible to identify genes that are associated with the target gene. Furthermore, a specific pathway can be dissected by treating cells sequentially with siRNAs targeting the various genes in the pathway and assaying which genes are affected. This will make it possible to assign a position in the pathway for each gene.
Gene Redundancy In many cases, eliminating the expression of a single gene in higher eukaryotes can be tolerated even if that gene product functions in a critical pathway. This is because many critical cell functions are accomplished by more than one gene product. When one gene product is eliminated, the redundant gene product compensates to allow the cell or animal to survive. Identifying redundant genes could be achieved by co-transfecting siRNAs and assaying for a given phenotype. Evaluating each of the candidate genes alone to ensure that they only cause the cell cycle defect when reduced in combination with the target gene would help pinpoint the most likely redundant gene
Functional Screening Libraries of siRNAs targeting broad collections of genes will enable screening experiments to tie genes to cellular function. To date, libraries with more than a couple of hundred siRNAs have been limited to a few large research organizations. Recognizing the benefits of siRNA libraries, Ambion is preparing a collection of more than 1800 siRNAs targeting the known human kinases. There have been no published reports on the application of siRNA libraries in screening experiments, but screens in Drosophila and C. elegans using dsRNA libraries exemplify the opportunities.
Biotechnology & Agriculture RNA interference has been used for applications in biotechnology, particularly in the engineering of food plants that produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase, a key enzyme in gossypol production, without affecting the enzymes production in other parts of the plant, where gossypol is important in preventing damage from plant pests.
Biotechnology & Agriculture… Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants. Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants and decreased the precursors of likely carcinogens in tobacco plants. Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy, resistance to common plant viruses, and fortification of plants such as tomatoes with dietary antioxidants.
C o n c l u s io n RN A i (R N A In t e r f e r e n c e )
RNA interference characteristics dsRNA needs to be directed against an exon, not an intron in order to be effective Homology of the dsRNA and the target gene/mRNA is required Targeted mRNA is lost (degraded) after RNAi The effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism) ssRNA does not work as well as dsRNA
Advantage of RNAi Downregulation of gene expression simplifies "knockout" analysis. Easier than use of antisense oligonucleotides. siRNA more effective and sensitive at lower concentration. Cost effective High Specifity middle region 9-14 are most sensitive With siRNA, the researcher can simultaneously perform experiments in any cell type of interest Can be labelled Ease of transfection by use of vector
Importance of RNAi Powerful for analyzing unknown genes in sequenced genomes. ⇒ efforts are being undertaken to target every human gene via siRNAs Faster identification of gene function Gene therapy: down-regulation of certain genes/ mutated alleles Cancer treatments knock-out of genes required for cell proliferation knock-out of genes encoding key structural proteins Agriculture