Thanks

1. 
1

2. -Sapate P D,
Student(ABW/34/2011),
Lokmangal Agril biotech college,
Wadala.
2

3. 
 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
3

4. 
 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.
4
After 45 days….

5. 
General outline
5

6. 
 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
6

7. 
 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
7

8. 
 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.
8

9. 
 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
9

10. 
HOW VIRUS REPLICATE ?
10

11. 
 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
11

12. 
 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.
12

13. 
 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.
13

14. 
 Unique DNA sequence
 Efficient cellular uptake
 Minimal nonspecific binding
 Target specific hybridization
 Non-toxic antisense construct
14
Characteristics of antisense
oligonucleotides

15. 
 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.
15
Approaches

16. 
 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.
16
1st approach

17. 
 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.
17
2nd approach

18. 
 They also show effective translational arrest .
 But the major problem is that they are generally
required higher molar concentrations than those
which activate RNaseH.
18

19. 
 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.
19
3rd approach

20. 
 They may form hybrids of
oligodeoxyribonucleotides & Oligoribonucleotides.
 The antiviral activity of an antisense oligonucleotides
depends usually on specific binding to a target
nucleic acid.
20

21. 
Over view 21

22. 
 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.
22
Ribozymes

23. 
23
Mechanism of ribozyme

24. 
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
24

25. 
25
Antisense as drug

26. 
 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
26

27. 
 Attempts are made to genetically engineer transgenic
plants to express antisense RNA instead activate the
RNAi pathway, although the processes result in
“gene silencing”.
27

28. 
 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 :-
28

29. 
Queries ?
(If any)
29

30. 
THANKs FOR YOUR
KIND ATTENSION
30