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  • http://www.rna.com/
  • www.rna.com
  • http://www.rna.com/
  • http://www.cambridge.org/catalogue/catalogue.asp?isbn=0511081316
  • http://www.cambridge.org/catalogue/catalogue.asp?isbn=0511081316
  • (1) http://www.youtube.com/watch?v=A-l8tqjm4Vg&feature=related(2) http://www.youtube.com/watch?v=3kdhYCJFmZc&feature=related
  • (1)*Meister ,G., Tuschl ,T.. (2004). Mechanisms of gene silencing by double-stranded RNA. Natural. 431(7006). 343-9.http://arabidopsis.info/students/rohan/mechanismrnai.html
  • http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • http://www.youtube.com/watch?v=kCxQdXX0Dbk
  • (1)*Meister ,G., Tuschl ,T.. (2004). Mechanisms of gene silencing by double-stranded RNA. Natural. 431(7006). 343-9.http://arabidopsis.info/students/rohan/mechanismrnai.html
  • (2)*Kedde ,M., Strasser ,M.JE., Boldajipour ,B., Oude Vrielink ,J.A., Slanchev ,K., le Sage ,C., Nagel ,R., Voorhoeve ,P.M., van Duijse ,J., Ørom ,U.A., Lund ,A.H., Perrakis ,A., Raz ,., Agami ,R.. (2007). RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. cell. 131(7). 1273-86.
  • (3)*Klionov ,M.S., Stoliarenko ,A.D., Riazanskiĭ ,S.S., Sokolova ,O.A., Konstantinov ,I.N., Gvozdev ,V.A.. (2007). Role of short RNAs in regulating the expression of genes and mobile elements in germ cells. ONTOGENES. 38(3). 213-27.
  • http://rnajournal.cshlp.org/content/13/3/422.full
  • http://rnajournal.cshlp.org/content/early/2007/01/19/rna.348307.full.pdf
  • (4)*Aalto ,A.P., Sarin ,L.P., van Dijk ,A.A., Saarma ,M., Poranen ,M.M., Arumäe ,U., Bamford ,D.H.. (2007). Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA(New York .N.Y.). 13(3), 422-9.
  • http://www.epibio.com/pdfforum/9_3dsrnarnaseiii.pdf
  • http://www.youtube.com/watch?v=h1kayIVEfcY&feature=related
  • http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Companies/RNAi_Therapeutics/index.html
  • http://www.alnylam.com/Programs-and-Pipeline/Programs/Liver-Cancer.php
  • http://www.alnylam.com/Programs-and-Pipeline/Programs/Hypercholestoralemia.php
  • http://www.alnylam.com/Programs-and-Pipeline/Programs/index.php(5)* DeVincenzo ,J., Cehelsky ,J.E., Alvarez ,R., Elbashir ,S., Harborth ,J., Toudjarska ,I., Nechev ,L., Murugaiah ,V., Van Vliet ,A., Vaishnaw ,A.K., Meyers ,R.. (2007). Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antivairal research . 77(3). 225-31.
  • http://www.proteigene.com/ecatalog/in-vivo-rna-lancer-4-injections-p-2172.html
  • DualChip® human RNAi side effect

(Rn ai) (Rn ai) Presentation Transcript

  • RNA interference GHAZWA SHAWASH
  • what is RNA?
  • * Why we prefer to work with RNA on human? Because DNA is traps inside the nucleus and can not go out and make works but RNA do that , so DNA is the source of information but nothing can happen without RNA , if we work on DNA we may case a mutation that will continue for ever .
  • RNA RNA technology is one of the most modern technologies that all the word goes toward it, but why is that? Because scientist have their vision witch http://www.rna.com/ is
  • strategy http://www.rna.com/
  • RNA R&D : * Vaccine Development * Cancer Diagnosis Kit * Functional Food and Feed * Genetic Engineering Vector System. * Virus Disease Therapeutics * Cancer Therapeutics http://www.rna.com/
  • RNA interference technology
  • RNA interference technology * RNA Interference (RNAi) technology has rapidly become one of the key methods used in functional genomics. http://www.cambridge.org/catalogue/catalogue.asp?isbn=0511081316
  • RNA interference technology * RNAi is used to block the expression of genes and create phenotypes that can potentially yield clues about the function of these genes. In the postgenomic era, the elucidation of the physiological function of genes has become the rate-limiting step in the quest to develop ‘genebased drugs’ and RNAi could potentially play a pivotal role in the validation of such novel drugs. * one of the winners of the 2006 Nobel Prize for the discovery of RNA Interference is Andrew Fire . http://www.cambridge.org/catalogue/catalogue.asp?isbn=0511081316
  • RNA interference technology * RNAi is a fantastic discovery , all the RNAi idea will be describing in these video. http://www.youtube.com/watch?v=H 5udFjWDM3E&feature=related (1) http://www.youtube.com/watch?v=A-l8tqjm4Vg&feature=related (2) http://www.youtube.com/watch?v=3kdhYCJFmZc&feature=related
  • The Mechanism of RNA Interference
  • The Mechanism of RNA Interference * The process by which RNAi occurs in different organisms varies slightly ; This is usually because different proteins are used in the process of RNAi . (1)*Meister ,G., Tuschl ,T.. (2004). Mechanisms of gene silencing by double-stranded RNA. Natural. 431(7006). 343-9. http://arabidopsis.info/students/rohan/mechanismrnai.html
  • The Mechanism of RNA Interference * Long double-stranded RNAs (dsRNAs; typically >200 nt) can be used to silence the expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, and plants). http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • The Mechanism of RNA Interference • Upon introduction, the long dsRNAs enter a cellular pathway that is commonly referred to as the RNA interference (RNAi) pathway. First, the dsRNAs get processed into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase IIIlike enzyme called Dicer (initiation step). This processing requires the presence of ATP http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • The Mechanism of RNA Interference * Then, the siRNAs assemble into endoribonuclease-containing complexes (multiprotein complex) known as RNA-induced silencing complexes (RISCs), unwinding in the process.(ATP-dependent step that activates the unwinding of the double stranded siRNAs ) . http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • The Mechanism of RNA Interference * The siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. (An endoribonuclease then cleaves this RNA, which is then thought to be degraded by exoribonucleases ) http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • The Mechanism of RNA Interference overall http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • The Mechanism of RNA Interference * In mammalian cells, introduction of long dsRNA (>30 nt) initiates a potent antiviral response, exemplified by nonspecific inhibition of protein synthesis and RNA degradation. The mammalian antiviral response can be bypassed, however, by the introduction or expression of siRNAs. http://www.ambion.com/techlib/append/RNAi_mechanism.html
  • This video for more understanding http://www.youtube.com/watch?v =kCxQdXX0Dbk http://www.youtube.com/watch?v=kCxQdXX0Dbk
  • types of small RNA's * There are three different types of small RNA's that occur naturally: (1) small interfering RNAs (siRNAs). (2) microRNAs . (3) repeat -associated short interfering RNAs (rasiRNAs). (1)*Meister ,G., Tuschl ,T.. (2004). Mechanisms of gene silencing by double-stranded RNA. Natural. 431(7006). 343-9. http://arabidopsis.info/students/rohan/mechanismrnai.html
  • microRNAs * MicroRNAs (miRNAs) are inhibitors of gene expression capable of controlling processes in normal development and cancer as an example . In mammals, miRNAs use a seed sequence of 6-8 nucleotides (nt) to associate with 3' untranslated regions (3'UTRs) of mRNAs and inhibit their expression. * We hypothesized that conserved regions in mRNAs may serve as docking platforms for modulators of miRNA activity (2)* Kedde ,M., Strasser ,M.J., Boldajipour ,B., Oude Vrielink ,J.A., Slanchev ,K., le Sage ,C., Nagel ,R., Voorhoeve ,P.M., van Duijse ,J., Ørom ,U.A., Lund ,A.H., Perrakis ,A., Raz ,E., Agami ,R.. (2007). RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. cell. 131(7). 1273-86.
  • Differences between siRNA and microRNA MicroRNAs predominantly suppress the translation of mRNA targets, while siRNAs not only prevent mRNA translation and/or lead to mRNA degradation, but are also involved in the regulation of gene expression at the transcriptional level. (3)*Klionov ,M.S., Stoliarenko ,A.D., Riazanskiĭ ,S.S., Sokolova ,O.A., Konstantinov ,I.N., Gvozdev ,V.A.. (2007). Role of short RNAs in regulating the expression of genes and mobile elements in germ cells. ONTOGENES. 38(3). 213-27.
  • producing long dsRNAs Current methods for producing long dsRNAs rely on annealing two single-stranded RNA (ssRNA) strands. We have tow methode : (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRp) of bacteriophage ϕ6 (Makeyev and Bamford 2000a,b) to generate dsRNA molecules. (2) an in vivo RNA replication system based on carrier state bacterial cells containing the ϕ6 polymerase complex (Onodera et al. 1992; Sun et al. 2004) to produce virtually unlimited amounts of dsRNA. Furthermore, we demonstrate the utility of this system by RNA knockdown experiments in two different cell types. http://rnajournal.cshlp.org/content/13/3/422.full
  • http://rnajournal.cshlp.org/content/early/2007/01/19
  • producing long dsRNAs In 2009 an easy and simple method to make RNAi constructs using two long oligonucleotides consisting of partially complementary sequences without the need for PCR amplification (polymerase chain reaction ) and multiple cloning steps . (4)*Aalto ,A.P., Sarin ,L.P., van Dijk ,A.A., Saarma ,M., Poranen ,M.M., Arumäe ,U., Bamford ,D.H.. (2007). Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA(New York .N.Y.). 13(3), 422-9.
  • Production of Short Double-Stranded RNA Simple , by Using RNase III . http://www.epibio.com/pdfforum/9_3dsrnarnaseiii.pdf
  • RNA i This vedio is for one of the scientests who discover the RNA Interference talking a bout RNAi technology . http://www.youtube.com/watch?v=h1kayIVE fcY&feature=related http://www.youtube.com/watch?v=h1kayIVEfcY&feature=related
  • RNAi Therapeutics Understudying http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Companies/RNAi_Therapeutics/index.html
  • Liver Cancers ALN-VSP targets two key genes each involved in the disease pathway of liver cancer: kinesin spindle protein, or KSP is involved in cancer proliferation, and vascular endothelial growth factor, or VEGF, is involved in cancer angiogenesis We believe that a dual-target approach in cancer increases the potential for a significant therapeutic benefit. In late 2008, an investigational new drug (IND) application for ALN-VSP was submitted to the U.S. FDA. http://www.alnylam.com/Programs-and-Pipeline/Programs/Liver-Cancer.php
  • Hypercholesterolemia ALN-PCS works by silencing the gene proprotein convertase subtilisn/kexin type 9, or PCSK9, which human genetics studies indicate to be an ideal target to treat hypercholesterolemia. Individuals with a genetically increased PCSK9 activity were found to have increased levels of LDL cholesterol, or bad cholesterol, together with an increased risk of cardiac disease. In contrast, individuals with genetically less PCSK9 activity were found to have significantly lower levels of LDL cholesterol and a significantly reduced risk of cardiac disease. Because of its novel mechanism of action, we believe that ALN-PCS has the opportunity to treat hypercholesterolemia in a way that is unaddressable by current therapies, such statin drugs. http://www.alnylam.com/Programs-and-Pipeline/Programs/Liver-Cancer.php
  • Respiratory Syncytial Virus Infection Respiratory syncytial virus infection, or RSV, is a serious respiratory infectious disease and the leading cause of pediatric hospitalizations in the U.S. Our RNAi therapeutic was designed to target the nucleocapsid "N" gene of the RSV genome, a gene that is required for the replication of the RSV. ALN-RSV01 silences the N gene, thereby reducing the virus' ability to reproduce. Our extensive pre-clinical work demonstrated potent and highly specific anti-viral efficacy with molecular proof of an RNAi mechanism of action. * It is a promising therapeutic candidate for further clinical development . (5)* DeVincenzo ,J., Cehelsky ,J.E., Alvarez ,R., Elbashir ,S., Harborth ,J., Toudjarska ,I., Nechev ,L., Murugaiah ,V., Van Vliet ,A., Vaishnaw ,A.K., Meyers ,R.. (2007). Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antivairal research . 77(3). 225-31. http://www.alnylam.com/Programs-and-Pipeline/Programs/Liver-Cancer.php
  • In Vivo RNA LANCEr, 4 injections MaxSuppressor™ In Vivo RNA-LANCEr uses a novel nontoxic blend of fat-soluble naturally-occurring hydrophobic molecules to efficiently deliver siRNA and miRNA into mouse organs. The advanced formulation enables users to to deliver RNAi agents, in vivo, for target validation and drug target identification purposes. After formulating RNAi agents with MaxSuppressor™ In Vivo RNA-LANCEr, the RNA becomes stable in serum, preventing the need for stability modifications which reduces the synthesis cost. www.proteigene.com/ecatalog/in-vivo-rna-lancer-4-injections-p-2172.html
  • RNAi TECHNOLOGY SIDE EFFECT human RNAi side effect : 1- Negative hybridization control probes (non specific DNA). These controls will be used to check for unspecific background. 2- Negative detection control spots (spotting solution). These controls will be used to check for unspecific background. 3- Positive biotin detection control spots. These controls will be used to check the fluorescence detection step in indirect labeling method. Different concentrations of positive biotin detection control are spotted on the last row of the microarray. DualChip® human RNAi side effect
  • THE END
  • References http://www.rna.com/ http://www.cambridge.org/catalogue/catalogue.asp?isbn=0511081316 http://www.youtube.com/watch?v=H5udFjWDM3E&feature=related http://www.youtube.com/watch?v=kCxQdXX0Dbk http://arabidopsis.info/students/rohan/mechanismrnai.html http://www.youtube.com/watch?v=h1kayIVEfcY&feature=related * Simple, Efficient Production of Short Double-Stranded RNA Using RNase III (Judith E. Meis, EPICENTRE).website : http://www.epibio.com/pdfforum/9_3dsrnarnaseiii.pdfmicroRNA formation and function http://www.youtube.com/watch?v=_-9pROnSD-A http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Companies/RNAi_Therapeutics/index.html http://www.alnylam.com/Programs-and-Pipeline/Programs/Liver-Cancer.php (1)* Meister ,G., Tuschl ,T.. (2004). Mechanisms of gene silencing by double-stranded RNA. Natural. 431(7006). 343-9. (2)* Kedde ,M., Strasser ,M.J., Boldajipour ,B., Oude Vrielink ,J.A., Slanchev ,K., le Sage ,C., Nagel ,R., Voorhoeve ,P.M., van Duijse ,J., Ørom ,U.A., Lund ,A.H., Perrakis ,A., Raz ,E., Agami ,R.. (2007). RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. cell. 131(7). 1273-86. (3)*Klionov ,M.S., Stoliarenko ,A.D., Riazanskiĭ ,S.S., Sokolova ,O.A., Konstantinov ,I.N., Gvozdev ,V.A.. (2007). Role of short RNAs in regulating the expression of genes and mobile elements in germ cells. ONTOGENES. 38(3). 213-27. (4)*Aalto ,A.P., Sarin ,L.P., van Dijk ,A.A., Saarma ,M., Poranen ,M.M., Arumäe ,U., Bamford ,D.H.. (2007). Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA(New York .N.Y.). 13(3), 422-9. http://books.google.jo/books?id=bjAm2mTbnPoC&pg=PA56&lpg=PA56&dq=repeat+associated+short+interfering+RNAs+(rasiRNAs)&source=bl&ots=ii34nFhrYx&sig=ABKvNRISLOdkGX0zwC4sW0iqbU&hl=en&ei=SMXxSanVBcLm-Ab1wLidDw&sa=X&oi=book_result&ct=result&resnum=1#PPP5,M1
  • References (5)*DeVincenzo ,J., Cehelsky ,J.E., Alvarez ,R., Elbashir ,S., Harborth ,J., Toudjarska ,I., Nechev ,L., Murugaiah ,V., Van Vliet ,A., Vaishnaw ,A.K., Meyers ,R.. (2007). Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antivairal research . 77(3). 225-31. http://www.openbiosystems.com/RNAi/siRNA/ http://images.google.jo/imgres?imgurl=http://venturebeat.com/wp-content/uploads/2008/01/tacere-logo 150px.gif&imgrefurl=http://venturebeat.com/tag/invdelphi ventures/&usg=__hfqTDQXbGqa6tYpfK6Fpm_x6M8A=&h=63&w=150&sz=3&hl=en&start=13&um=1&tbnid=xr6GhM7WingFl M:&tbnh=40&tbnw=96&prev=/images%3Fq%3Drx%252Brnai%252Bdrug%26hl%3Den%26um%3D1 (6) DualChip® human RNAi side effect 1.1 Related topic: Transgenic ( Transgenic animals , Sonja Voss, Eppendorf AG, Hamburg, Germany ).