Antisense RNA technology

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WELCOME

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Antisense RNA technology

-Sapate P D,Student(ABW/34/2011),

Lokmangal Agril biotech college,Wadala.

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Antisense RNA is a single-stranded RNA that

is complementary to a messenger RNA (mRNA) strand transcribed within a cell

Antisense RNA introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and creating barrier to the translation machinery.

E.g. hok/sok system of the E. coli R1 plasmid.

INTRODUCTION

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This translational arrest causes reduced

amount of protein expression. Well-known examples of GM plants produced

by this technology- The Flavr Savr tomato , Two cultivars of ring spot-resistant papaya.

After 45 days….

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General outline

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The intended effect in both will be same i.e.

gene silencing but the processing is little but different.

Antisense technology degrades RNA by enzymes RNaseH while RNAi employed the enzyme DICER to degrade the m RNA.

RNAi are twice larger than the antisense oligonucleotide.

Diff. between antisense technology & RNAi

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First time at “ free university of Amsterdam”,

used antisense RNA technology against the gene determining flower color of petunia .

Antisense effect first demonstrated by zemencnick & Stephenson in 1970 on “Rous sarcoma virus”.

First time antisense oligonucleotides are synthesized by Eckstein and colleagues.

HISTORY

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In 1995 Guo and Kemp hues: injection of either antisense or sense RNAs in the germ line of C. elegans was equally effective at silencing homologous target genes.

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There is HOK(host killing)/SOK(suppress killing)

system in R1 plasmid in E.Coli. when E. coli cell undergoes division , daughter cell

inherit hok gene & sok gene from parent. But due to short life of cell, the sok gene is get degraded. So in normal cell, hok gene get over expressed & cell get die.

But when R1 plasmid is get inherited , it having the sok gene & sok promoter.

Then it transcripts sok gene & it is get overexpressed against hok gene.

Nature’s antisense system

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HOW VIRUS REPLICATE ?

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In this technique, Short segments of single

stranded RNA are introduced. These oligonucleotides are complementary to

the mRNA, which physically bind to the mRNA. So , they block the expression of particular

gene. In case of viruses, antisense oligonucleotides

inhibit viral replication with blocking expression of integrated proviral genes.

Usually consist of 15–20 nucleotides.

MECHANISM

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Translation of mRNA may be blocked by two

possible mechanisms , These are:- 1] by base specific hybridization – which prevents access by translation machinery i.e. “hybridization arrest”. 2] by forming RNA/DNA duplex which is recognized by nuclease RNaseH , specific for digesting RNA in an RNA/DNA duplex.

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RNaseH is a non-specific endonuclease,

catalyzes the cleavage of RNA via hydrolytic mechanism.

RNaseH has ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA duplex.

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Unique DNA sequence Efficient cellular uptake Minimal nonspecific binding Target specific hybridization Non-toxic antisense construct

Characteristics of antisense

oligonucleotides

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The antisense technology can be modified in

THREE modes because of chemical modifications of the oligonucleotides.

These modes are due to activation of RNaseH & internucleotides linkages which do not activate enzyme.

Approaches

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The antisense oligonucleotides binds the target

sequence causing both “hybridisation arrest ” & “RNaseH activation”. Degradation of mRNA by RNaseH results into

release of oligonucleotides. They may bind to other copies of target mRNA. These oligonucleotides are also susceptible to

other nucleases. This a major parameter affecting catalytic

mode of degradation.

1st approach

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In this, antisense oligonucleotides binds to

target sequence result in translation arrest but they do not activate enzyme RNaseH.

Oligoribonucleotides & there analogues , oligodeoxyribonucleotides , various non phosphate & phosphate internucleotides linkages fall in this category.

They show resistance against nucleuses enzyme and never get degraded by them.

2nd approach

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They also show effective translational arrest . But the major problem is that they are

generally required higher molar concentrations than those which activate RNaseH.

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It combines features of both previous approaches.

They contains both internucleotides linkages which are responsible for RNaseH activation & which shows resistance against them.

Digestion of mRNA target in RNA-DNA duplex releases oligonucleotides which are resistance against nuclease enzyme, hence are more effective than oligonucleotides in 1st approach.

3rd approach

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They may form hybrids of

oligodeoxyribonucleotides & Oligoribonucleotides.

The antiviral activity of an antisense oligonucleotides depends usually on specific binding to a target nucleic acid.

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Over view

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Thomas and coworkers coined the term

‘ribozymes’. These are RNA molecules which have catalytic

activity which degrade nucleotides . Ribozyme Bind to the target RNA moiety and

inactivate it by cleaving the phosphodiester backbone at a specific cutting site.

Ribozyme destroy RNA that carries the massage of disease.

These are effectively used against HIV virus.

Ribozymes

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Mechanism of ribozyme

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1. Flavr Savr tomato- antisense RNA used against an enzyme polygalacturonase, an softening enzyme which is responsible for ripening.

2. Transgenic ACMV-resistant cassava plants* – Used against African cassava mosaic virus (ACMV) which causes cassava mosaic disease causing major economic loss in Africa.

3. Formivirsen- is the first antiviral drug developed against CMV.

APPLICATION

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Antisense as drug

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Antisense technology shows potential for diverse

application to field of basic research & therapy. One of the most approved approaches for

inactivating a single specific gene. But it may sometime give undesirable effect. Generally , antisense RNA still lack effective design,

biological activity, and efficient route of administration.

Antisense technologies form a very powerful weapon for studying gene function and for discovering more specific treatments of disease.

conclusion

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Attempts are made to genetically engineer

transgenic plants to express antisense RNA instead activate the RNAi pathway, although the processes result in “gene silencing”.

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A textbook of biotechnology 2nd edition by H.

D. Kumar www.youtube.com Nature biotechnology. www.ncbi.nlm.nih.com (PubMed ID

17173627)* www.google.com

References :-

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Queries ? (If any)

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THANKs FOR YOUR KIND ATTENSION