Antisense RNA Technology

Prepared By:Priya Upadhyay

B.Tech(B.T)Vth semester

Submitted To:Dr. Sarika Gupta

http://www.thedailysheeple.com/wp-content/uploads/2015/05/DNA-Strand.jpg

CONTENTSIntroductionHistoryLevels of gene silencingTranscriptional gene silencingPost transcriptional gene silencingSalient features of RNAiMechanism of RNAiApplicationsAdvantages and disadvantagesFuture perspectivesCase studyConclusionReferences

http://www.jurassicworld.com/media/creation-lab/mrdna/2-sequencing-mr-dna.png

INTRODUCTIONWHAT IS ANTISENSE RNA TECHNOLOGY?

Antisense RNA Technology > tool used for inhibition of gene expression.

In this technique Short segments of single stranded DNA called oligo de oxy nucleotides are introduced.

These oligonucleotides are complementary to the mRNA, which physically bind to the mRNA.

This technique prevents the synthesis of specific protein.

Powerful weapon for Studying gene function and for discovering more specific treatments of diseases.

Gene silencing =Antisense RNA technology = Ribozyme technology.https://lh6.ggpht.com/unnamed.jpg

HISTORY 1990- Richard Jorgenson gave the phenomenon of

‘cosupression of gene expression’.

1992- Romano and Macino gave the phenomenon of as Virus Induced Gene Silencing , set of such phenomenon was termed as post transcriptional gene silencing.

1995-RNA silencing was first documented in animals by Guo and Kemphues, and for this phenomenon they coined the term antisense mediated silencing.

1998-Andrew Fire and Craig C.Mello coined the term RNA interference (RNAi) .

http://bgiamericas.com/wp-content/uploads/2011/08/Cropped-DNA-01.jpg

2001-Thomas Tuschl, discovered with his colleagues that RNAi could be prompted through the use of shorter pieces of RNA known as small interfering RNAs (siRNAs).

2001-Gregory Hannon identified, described, and named the "Dicer" enzyme, which chops dsRNA into siRNAs, as well as the RNA-induced silencing complex (RISC), which mediates the silencing process by degrading the homologous mRNA7.

2002-Effect named "short hairpin-activated gene silencing" or SHAGging was introduced.

2004 Morris et al. Observed that siRNA silences genes at the transcriptional level.

History Contd….

http://bgiamericas.com/wp-content/uploads/2011/08/Cropped-DNA-01.jpg

LEVELS OF GENE SILENCING 1. Transcriptional gene silencing (TGS) It Causes gene silencing by: • DNA methylation. • Heterochromatin formation. • Programmed DNA elimination. 2. Post transcriptional gene silencing

(PTGS) It is known commonly as RNA

interference(RNAi). It causes silencing by

destruction ofthe mRNA of the gene to which the

siRNAshows perfect complementarity.

http://cdn01.wallconvert.com/_media/wallpapers_1680x1050/1/1/dna-strand-3111.jpg

TRANSCRIPTIONAL GENE SILENCING Transcriptional gene silencing - Result of modifications of either the histones or DNA.Gene silencing by modification of Histones and DNA Modification of nucleosomes alter the accessibility of

the gene to the transcriptional machinery and regulatory proteins

Heterochromatin is commonly involved in gene silencing, and affects large sections of DNA.

Methylation of particular DNA sequences can also

silence transcription in many eukaryotes.

https://lh6.ggpht.com/unnamed.jpg

POST TRANSCRIPTIONAL GENE SILENCING

RNA interference (RNAi) is a molecular mechanism in

which fragments of double stranded nucleic acid (dsRNA)

interfere with the expression of a particular gene .

The dsRNA can be either, MicroRNA (miRNA) or Small

interference RNA (siRNA)

https://lh6.ggpht.com/unnamed.jpg

SALIENT FEATURES OF MICRO RNA

Double stranded RNA rather than single–stranded antisense RNA is the interfering agent.

High degree of specific gene silencing with less effort.

Highly potent and effective, only a few double stranded RNA molecules per cell are required for effective interference.

Silencing can be introduced in different developmental stages

Systemic silencing Avoids problems with abnormalities caused by a

knocked out gene in early stages which could mask desired observations.

Silencing effects passed through generations https://lh6.ggpht.com/unnamed.jpg

MECHANISM OF RNAIInitiation step:1.Double stranded RNA(dsRNA) molecule is cleaved to form 21-23 bp double stranded fragments called short interfering RNAs(siRNAs).

Effector step:1.siRNA is unbound by helicase activity associated with a multiprotein complex known as RNA-induced silencing complex(RISC).

2.The antisense RNA complexed with RISC binds to its corresponding mRNA which is cleaved by the enzyme Slicer rendering it inactive.

http://www.scq.ubc.ca/wp-content/siRNA.gif

SIGNIFICANCE AND APPLICATIONS

In biological functions >immunity >downregulation of genes

>upregulation of genes >evolution

In technology >gene knockdown >functional genomics

>medicines >biotechnology

http://www.jurassicworld.com/media/creation-lab/mrdna/3-assembly-mr-dna.png

ADVANTAGES AND DISADVANTAGES

This process is able to affect only selected genes which the RNAi is complementary to.RNAi will bind to most of the complementary genes it encounters, making it highly efficient as well as robustShown to work in the laboratory and shows potential in mammalian cellAble to be used on a large scale

Delivery, i.e., getting those exquisitely specific siRNAs to the appropriate sites in the appropriate amounts to ensure appropriate uptake and the intended silencing remains a considerable challenge Off-target effects i.e., when siRNA can affect unintended genes in the organism which may be vital

advantages disadvantages

http://bgiamericas.com/wp-content/uploads/2011/08/Cropped-DNA-01.jpg

FUTURE PERSPECTIVES Since 1998 ,RNAi discovery has been touted

as a technical breakthrough in biological research.

Even with RNAi's rapid development over the years, it is still in its infancy stage. A better understanding of the mechanisms that take place will help reduce problems such as off-target effects.

In 2001 RNAi was used to treat hepatitis in mice

With further knowledge about the mechanisms of RNAi it may be the gateway for other emerging technologies such as transgenic studies, gene therapy and gene-wide screening..

Whilst still in process, it opens the doors of what can be achieved, and infact realises a small part of the hope - that nothing is untreatable.

http://www.godandscience.org/images/dna-helix.gif

CASE STUDY- FLAVR SAVR TOMATO

The first FDA approved genetically modified food

Licensed in 1994 will not soften while ripening on the vine. Increased shelf life , tomatoes can be

shipped safely, keep their color, and have their natural flavors.

BEFORE THE FLAVR SAVR Picked before ripe and gassed with

ethylene to give red color – keeps them from becoming spoiled

Tomatoes lose their taste and taste more like “cardboard.”

Flavr Savr Tomato developed by Calgene (Sources: http://www.ca.uky.edu/agripedia/glossary/flavr.htm)

MAKING OF FLAVR SAVR Enzyme Polygalacturonase

breaks down structural polysaccharide pectin in wall of a plant.

This is part of the natural decay process in a plant

Flavr savr tomatoes have been constructed that express an antisense mRNA complementary to mRNA for an enzyme involved in ethylene production

These tomatoes make only 10% of normal amount of enzyme thus delaying ethylene production.

http://www.google.com/imgres?q=Flavr+Savr+Antisense technology

GM VS. TRADITIONAL TOMATOFlavr Savr Tomato Traditional Tomato

The Flavr Savr tomato ripens on the vine – resulting in fuller flavor. It is modified so that it remains firm after harvesting

The traditional tomato must be harvested while it is still green and firm so that it is not crushed on the way to the supermarket.

The traditional tomato is sprayed with ethylene after shipping to induce ripening.

Ripe and Increased Flavor.

Ripe but decreased flavor.

Supermarket

Flavr Savr is modified tomato for suiting modern productions and distributions. Credit: Owen Koo

OUTCOMES: Basically, the gene in the

tomato stops the tomato from softening during ripening so that it is easier to ship but keeps its natural flavors too.

The tomato also has a much longer shelf life and keeps from spoiling quickly.

PROBLEMS WITH FLAVR SAVR: Safety- health risks, some

environmental risks Possible monopolies for

businesses Ethical concerns Only rich countries can afford it

http://www.jurassicworld.com/media/creation-lab/mrdna/1-extraction-mr-dna.png

CONCLUSION…The Antisense RNA technology shows the potential fordiverse applications to basic research and therapy.

Antisense technology offers almost unlimited scope for

the development of new methods of drug design and one

of the most approved approaches among several others,

for inactivating a single chosen gene.

However, the full commitment of this promise is yet to be

established.

http://blogs-images.forbes.com/daviddisalvo/files/2011/11/DNA.jpg

REFERENCESSyed M Shah, N Saini , S Ashraf, G Ravi Kumar, ‘Gene Silencing, Mechanism and Applications ’,DHR International Journal Of Biomedical and Life Sciences (DHR-IJBLS) ISSN: 2278-8301, Vol. 3(1), 2012Stuti Gupta, Ravindra Pal Singh, Nirav Rabadia, Gaurang Patel, Hiten Panchal, ‘ANTISENSE TECHNOLOGY’, Volume 9, Issue 2, July – August 2011; Article-007, ISSN 0976 – 044Xhttps://cellbiology.med.unsw.edu.au/cellbiology/index.php?title=Group_2_Project_RNA_Interference&oldid=2374Lu Tian,(November 25, 2014),’ The Flavr Savr Tomato’, www.slideshare.comKirti Khuntwal,’gene silencing’, www.authorstream.comAshok Singamsetti, 19th April, 2014,’Transgenics Production and Bio-Safety concerns’, Department of Genetics and Plant BreedingGeorge L. Sen and Helen M. Blau,’A brief history of RNAi: the silence of the genes’, doi: 10.1096/fj.06-6014rev July 2006 The FASEB Journal vol. 20 no. 9 1293-1299

QUERIES…??