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RNAi   Overview http://www.nature.com/nrg/multimedia/rnai/animation/index.html RNAi   Overview http://www.nature.com/nrg/multimedia/rnai/animation/index.html

RNAi Overview http://www.nature.com/nrg/multimedia/rnai/animation/index.html - PowerPoint Presentation

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RNAi Overview http://www.nature.com/nrg/multimedia/rnai/animation/index.html - PPT Presentation

What is RNAi RNA interference RNAi is a natural process that cells use to turn off or silence unwanted or harmful genes The initial discovery of this phenomenon was in 1991 by scientists trying to deepen the ID: 652114

rna rnai cells sirna rnai rna sirna cells gene strand genes interference short dsrna cell protein sequence double cleavage

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Presentation Transcript

Slide1

RNAi Overview

http://www.nature.com/nrg/multimedia/rnai/animation/index.htmlSlide2

What is RNAi

?

RNA interference (RNAi) is a natural process that cells use to 'turn off' or silence unwanted or harmful genes. The initial discovery of this phenomenon was in 1991, by scientists trying to deepen the

colour

of petunias. Surprisingly, by introducing a gene for

colour

, they found that they had turned off the gene.  Slide3

What is RNAi

?

Several years after the petunia experiments, the mechanism of RNA interference was revealed: it is triggered by double-stranded RNA (dsRNA), not usually found in healthy cells, but needed to turn genes off, if the cell is threatened or damaged by invading viruses.Slide4

THE PROCESS

In natural RNA interference, dsRNA in the cell’s cytoplasm is cut by an enzyme called Dicer into double stranded small interfering RNA (siRNA) molecules which are 20-25 nucleotides long.

This siRNA binds to an RNA-Induced Silencing Complex (RISC) which separates the two strands into the passenger and guide strand.

The passenger strand is degraded while the RISC takes the guide strand to a specific mRNA site, cleaving it so that the unwanted target protein is not produced.

This is how the gene is ‘silenced’.Slide5

A brief history of RNA interference (RNAi)Slide6

RNA Interference (RNAi) is one of the most important technological breakthroughs in modern biology, allowing us to directly observe the effects of the loss of function of specific genes in mammalian systems.

In the early 1990s, a number of scientists observed independently that RNA inhibited protein expression in plants and fungi.

This phenomenon, identified but not understood, was then known as “posttranscriptional gene silencing” and “quelling”.

In 1998 Fire and Mello observed in

Caenorthabditis

elegans

that double-stranded RNA (dsRNA) was the source of sequence-specific inhibition of protein expression, which they called “RNA interference”. 

While the studies in C.

elegans

were encouraging at that time the use of RNAi as a tool was limited to lower organisms because delivering long dsRNA for RNAi was nonspecifically inhibitory in mammalian cells. 

Fire and Mello won the 2006 Nobel Prize in Physiology or Medicine for their discovery of RNA interference.Slide7

Further studies in plants and invertebrates demonstrated that the actual molecules that led to RNAi were short dsRNA oligonucleotides, 21 nucleotides in length, processed internally by an enzyme called

“Dicer”.

 

The Dicer cleavage products were referred to as

“short interfering RNA”

now popularly known as

“siRNA”.

 

Subsequently in 2001, it was demonstrated that siRNA could directly trigger RNAi in mammalian cells without evoking nonspecific effects.

Today we have a greater understanding of the components that are part of the RNAi pathway, the efficiency with which these components function, the specificity of sequence recognition and cleavage of cellular mRNA and many of the key requirements for designing and generating extremely effective RNAi reagents. 

Our analysis of novel chemistries will further improve this approach and lead the way to in vivo analysis and potentially make the use of RNAi in therapeutics possible.Slide8

Applications of RNAi

RNAi technology can be used to identify and functionally assess the thousands of genes within the genome that potentially participate in disease phenotypes.

In addition, RNAi technology provides an efficient means for blocking expression of a specific gene and evaluating its response to chemical compounds or changes in signaling pathways.

Small (or short) interfering RNA (siRNA) is the most commonly used RNA interference (RNAi) tool for inducing short-term silencing of protein coding genes. siRNA is a synthetic RNA duplex designed to specifically target a particular mRNA for degradation.

While siRNA provides the opportunity to induce gene knockdown in a variety of cell lines, their utility is limited to cells that are amenable to transfection of synthetic oligonucleotides. Since siRNAs achieve transient silencing, experiments are limited to relatively short time frames on the order of 2-4 days. Slide9

How does

siRNA

work?

siRNAs consist of two RNA strands, an antisense (or guide) strand and a sense (or passenger) strand, which form a duplex 19 to 25

bp

in length with 3' dinucleotide overhangs .

Small double-strand siRNAs are transfected into cells where the guide strand is loaded into RISC. This activated protein and nucleic acid complex can then elicit gene silencing by binding, through perfect complementarity, to a single target mRNA sequence, thereby targeting it for cleavage and degradation.Slide10

How is siRNA delivered to a cell?

siRNA must be transfected into cells either by cationic lipid or polymer-based transfection reagents, electroporation (physical delivery via plasma membrane holes created by an electrical field), or adding chemical modifications to the duplex to aid in uptake by the cell.Slide11

Applications

siRNAs are widely used to assess the individual contributions of genes to an assortment of cellular phenotypes including cytokinesis, apoptosis, insulin signaling and cell differentiation .

siRNA screens have been used to identify novel pathways and have had significant impact in validating targets for a number of cellular processes and diseases including cancer , HIV infection and hepatitis

Finally, 

in vivo

 RNAi has been used for target validation studies in animal disease models and has the potential to be used for therapeutic purposes where disease-causing genes are selectively targeted and suppressed.Slide12

Glossary

CLEAVAGE:

 The splitting of a large or complex molecule into smaller or simpler molecules.

PATHWAY: a sequence of reactions, usually controlled and catalyzed by enzymes, by which one organic substance is converted to another.