Antisense technology uses short DNA sequences called oligonucleotides that are complementary to messenger RNA (mRNA) to prevent specific proteins from being synthesized. When introduced into cells, these antisense oligonucleotides bind to their target mRNA through Watson-Crick base pairing, forming RNA-DNA hybrids that are degraded by RNase H enzyme. This prevents translation and expression of the target protein. There are three generations of antisense oligonucleotides that have been developed with improved stability and targeting capabilities, including phosphorothioate, 2'-O-methyl RNA, and locked nucleic acid chemistries. Antisense technology has potential applications in treating diseases like cancer, viral infections, and genetic disorders.

1. ANTISENSE TECHNOLOGY
Presented By
Desh Bandhu Gangwar
M.Tech Biotech (2 year)
Concerned Faculty
Dr. Gunjan Garg
Assistant Professor
School of Biotechnology

2. INTRODUCTION
What is Antisense
Technology ?

4. 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.

5. 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.

6. Antisense Oligonucleotides
What are Antisense
Oligonucleotides?

7. 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.

8. When these AS-ON combined
with target mRNA, a DNA/RNA
hybrid form,which degraded by
the enzyme RNase H.
RNase H

9. 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.

10. Mechanism of antisense activity

11. Types Of AS-ON
First generation AS-ON
Second generation AS-ON
Third generation AS-ON

12. 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

13. 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.

14. Sites of chemical modification

15. 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.

16. 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.

17. 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.

18. 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.

19. 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)

20. 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

21. 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

22. 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.

23. Mechanism of Ribozymes

24. Types Of Ribozymes
Tetrahymena group I intron
 RNase P
Hammer head ribozyme
Hairpin ribozyme
Hepatitis delta virus ribozyme

25. Cycle of RNA cleavage by hammerhead ribozyme

26. Ribozymes in clinical trials
ANGIOZYME - VEGF-receptor1
HERZYME - HER-2
HEPTAZYM

27. 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.

28. Mechanism of RNA interference

29. Comparision Of different Antisense stratgies

30. Applications Of Antisense technologies
Story of Flavr Savr…

31. Antisense therapy
ß-thalassemia
Cytomegalovirus retinitis
Hemorrhagic fever viruses
Duchenne muscular dystrophy
Cancer
HIV/AIDS
High cholesterol

32. Antisense Drug Therapy

33. 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

34. QUERIES?

35. THANK YOU