ANTISENSE TECHNOLOGY

Presented By
Desh Bandhu Gangwar
M.Tech Biotech (2 year)

Concerned Faculty
Dr. Gunjan Garg
Assistant Professor
School of Biotechnology
 INTRODUCTION

What is Antisense
Technology ?
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.
Antisense technology prevent
the synthesis of specific protein.

Antisense technologies are a

suite of techniques that, together
form a very powerful weapon for
studying gene function and for
discovering more specific
treatments of disease.
Antisense Oligonucleotides

What are Antisense

Oligonucleotides?
The antisense effect of a
oligonucleotide sequence was
first demonstrated in 1970s by
Zamecnik and Stephenson, in
Rous sarcoma virus.

AS-ONs usually consist of 15–

20 nucleotides, which are
complementary to their target
mRNA.
When these AS-ON combined
with target mRNA, a DNA/RNA
hybrid form,which degraded by
the enzyme RNase H.

RNase H
RNase H is a non-specific
endonuclease, catalyzes the
cleavage of RNA via hydrolytic
mechanism.

RNase H has ribonuclease

activity cleaves the 3’-O-P bond
of RNA in a DNA/RNA duplex.
Mechanism of antisense activity
 Types Of AS-ON

First generation AS-ON

Second generation AS-ON

Third generation AS-ON

A successful AS-ON depends on the following
characteristics:

 Unique DNA sequence

 Efficient cellular uptake
 Minimal nonspecific binding
 Target specific hybridization
 Non-toxic antisense construct
 Nuclease resistant to protect AS-ON
 First generation AS-ON
Firstsynthesized by Eckstein and
colleagues.

Phosphorothioate - oligo deoxy

nucleotides are the major
representatives of first generation
DNA analogs that are the best
known.
Sites of chemical modification
Phosphorothioate linkages in Ons
primarily used to enhance their
nuclease resistance.

Inthis class of ONs, non bridging

oxygen atoms in phopho-diester
bond is replaced by sulfur.

They first used as AS-ONs for the

inhibition of HIV.
 Characterstics of first generation AS-ON

Better stability to nucleases but still

degrades.
Decreased affinity to target mRNA.
Enhanced specificity of hybridization.
Toxic in nature.
Can activate R Nase H.
 Second generation AS-ON

Second generation ONs

containing nucleotides with
alkyl modifications at the 2’
position of the ribose.

 2’-O-methyl and 2’-O-methoxy-

ethyl RNA are the most
important member of this class.
 Characterstics of second generation AS-ON

Best stability to nucleases.

Increased affinity to target mRNA.
Less toxic than first generation
AS-ON.
Can not activate R Nase.
 Third generation AS-ON
Newest and most promising.
Enhance binding affinity and
biostability.

Peptide nucleic acids (PNAs)

Locked nucleic acid (LNA)
Tricyclo-DNA (tcDNA)
Cyclohexene nucleic acids (CeNA)
Peptide nucleic acids

 In PNAs the deoxyribose phosphate

backbone is replaced by polyamide
linkages, which is composed of
repeating N-(2-aminoethyl)-glycine
units, linked by peptide bonds
 PNA was first introduced by Nielsen
and coworkers in 1991.
 They are electrostatically neutral
molecules
Locked nucleic acid

 LNA was synthesized by Jesper

Wengel in 1998.
 The ribose moiety of LNA nucleotide
is modified with an extra bridge
connecting the 2' oxygen and 4'
carbon
 Ribozymes
Thomas and coworkers coined the
term ‘ribozymes.
Ribozymes are RNA molecules
that have catalytic activity.
Ribozyme Bind to the target RNA
moiety and inactivate it by
cleaving the phosphodiester
backbone at a specific cutting
site.
Mechanism of Ribozymes
 Types Of Ribozymes

Tetrahymena group I intron

 RNase P
Hammer head ribozyme
Hairpin ribozyme
Hepatitis delta virus ribozyme
Cycle of RNA cleavage by hammerhead ribozyme
 Ribozymes in clinical trials

ANGIOZYME - VEGF-receptor1

HERZYME - HER-2

HEPTAZYM
 RNA interference
 RNA interference (RNAi) is a system
within living cells that takes part in
controlling genes activity.

 Twotypes of small RNA molecules –

(miRNA) and (siRNA) are central to
RNA interference.

 Melloand Fire named the process

RNAi, were awarded the Nobel Prize.
Mechanism of RNA interference
Comparision Of different Antisense stratgies
Applications Of Antisense technologies

Story of Flavr Savr…

 Antisense therapy

ß-thalassemia
Cytomegalovirus retinitis
Hemorrhagic fever viruses
Duchenne muscular dystrophy
Cancer
HIV/AIDS
High cholesterol
Antisense Drug Therapy
 REFERENCE
 Gene cloning and DNA analysis, Fifth edition
By T.A Brown Page no. 235
 Walton, S. P., Roth, C. M., Yarmush, M. L.
“Antisense Technology.”The Biomedical
Engineering Handbook: Second Edition.
 Indian journal of chemistry vol. 48 B
December 2009, pp. 1721-1726
 Indian journal of biotechnology vol 4,JUL
2005,pp. 316 -322
 Eur. J. Biochem. 270,1628–1644
 Clinical and Experimental Pharmacology and
Physiology (2006) 33, 533–540
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