(rn ai)

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 is

http://www.rna.com/

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


* 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 ‘gene-based 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 .


* RNAi is a fantastic discovery , all the RNAi idea will be describing in these video.

http://www.youtube.com/watch?v=H5udFjWDM3E&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 process by which RNAi occurs in different organisms varies slightly ; This is usually because different proteins are used in the process of RNAi .

The Mechanism of RNA Interference

(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


* 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


• 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 III-like enzyme called Dicer (initiation step).

This processing requires the presence of ATP



* 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 ) .



* 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 )


overall




* 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.


This video for more understanding

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., vanDuijse ,J., Ørom ,U.A., Lund ,A.H., Perrakis ,A., Raz ,E., Agami ,R.. (2007). RNA-binding protein Dnd1 inhibits microRNAaccess to target mRNA. cell. 131(7). 1273-86.

Differencesbetween 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://www.rna.com/


http://rnajournal.cshlp.org/content/early/2007/01/19/rna.348307.full.pdf

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=h1kayIVEfcY&feature=related




RNAi Therapeutics

Understudying

http://www.rnaiweb.com/RNAi/RNAi_Web_Resources/RNAi_Companies/RNAi_Therapeutics/index.html

http://www.alnylam.com/

Liver CancersALN-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

http://www.youtube.com/watch?v=aKBZbxBnpGM

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 un-addressable by current therapies, such statin drugs.


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.

In Vivo RNA LANCEr, 4 injections

MaxSuppressor™ In Vivo RNA-LANCEr uses a novel non-toxic 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://arabidopsis.info/students/rohan/mechanismrnai.html


* 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



(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=ABKvNRISLOdkGX0zwC4sW0i-qbU&hl=en&ei=SMXxSanVBcLm-Ab1wLidDw&sa=X&oi=book_result&ct=result&resnum=1#PPP5,M1

http://www.rna.com/







http://www.epibio.com/pdfforum/9_3dsrnarnaseiii.pdf

http://www.youtube.com/watch?v=_-9pROnSD-A



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

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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 ).

http://www.openbiosystems.com/RNAi/siRNA/

http://images.google.jo/imgres?imgurl=http://venturebeat.com/wp-content/uploads/2008/01/tacere-logo

(rn ai)

Technology

rna rna technology

rna interference technology

discovery of rna interference

rna degradation

rna rd

rna interference rnai

cleavage of cognate

complementary rna molecules