Antisense Technology, Ribozyme

1. gggdfdf

2. PRINCIPLES OF BIOTECHNOLOGY
MBB-501
Presented by submittedto
Viveksuthediya Mr.R.S. deshpande
Reg. no. Adpm/19/2680

3. CONTENTS
Introduction
History
Mechanism
Approaches
Application
Conclusion
References

4. Antisense Technology
 The tool that is used for the inhibition of gene
expression is called Antisense technology.
 The antisense technology uses following agents for
inhibition of gene expression.
I ) Antisense Oligonucleotides
II ) Ribozymes
III) Short Interfering RNA (siRNA)
IV) Micro RNA (miRNA)

6. Antisense Oligo-Nucleotides
Introduction :
 Antisense oligonucleotides is a single stranded RNA that is
complementary to a messenger RNA (mRNA) strand
transcribed in the cell.
 Antisense oligonucleotides introduced into a cell to inhibit
translation of a complementary mRNA by base pairing to it
and creating barrier to the translation machinery.
 This translation 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.

7. History :
 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
and Stephenson in 1970 on “Robus sarcoma virus”.
 First time antisense oligonucleotides are
synthesized by Eckstein and colleagues.

9. Sense Strand :
 It is the strand that is not transcribed into RNA.
 The base sequence of sense strand of a gene is the
same as that of the mRNA produced by it ( except
for T in the place of U ).
 Hence, the nhRNA/mRNA produced by a gene in
normal orientation is also known as sense RNA.
 It is oriented in 5’→3’ orientation.

10. Antisense Strand :
It is a strand that is transcribed into RNA.
 Its sequence is complementary to the mRNA
sequence .
 The antisense strand is also referred to as the
template strand.
 The oligonucleotide complementary to the mRNA
is called ‘antisense’ oligonucleotide.
 It is oriented in 3’→5’ direction.

11. Fig. Sense and Antisense Strand

12. Mechanism :
 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 a particular gene.
 Antisense oligonucleotides usually consists of 15-20
nucleotides.
 Translation of mRNA may be blocked by two possible
mechanisms :
I ) By base specific hybridization, which prevents translation
of mRNA.

13. II ) By forming RNA/DNA duplex which is recognized by
nuclease RNaseH, specific for digesting RNA in an
RNA/DNA duplex.
 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.

14. Characteristics of Antisense Oligonucleotides :
 Unique DNA sequence
 Efficient cellular uptake
 Minimal nonspecific binding
 Target specific hybridization
 Non-toxic antisense construct

15. Approaches :
 The antisense technology can be modified in three
modes because of chemical modifications of the
oligonucleotides.
 These modes are due to activation of RNaseH and
internucleotides linkages which do not activate
enzyme.

16. 1st Approach :
 The antisense oligonucleotudes binds the target sequence
causing both “hybridization arrest” and “RNaseH activation”.
 Degradation of mRNA by RNaseH results into release of
oligonuleotides.
 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.

18. 2nd Approach :
 In this, antisense oligonucleotides binds to target sequence
result in translation arrest but they do not activate enzyme
RNaseH.
 Oligoribonucleotides and analogues,
oligodeoxyribonucleotides, various non phosphate and
phosphate internucleotides linkages fall in this category.
 They show resistance against nucleases enzyme and never
get degraded by them.
 They also show effective translation arrest.
 But the major problem is that they are generally required in
higher molar concentrations than those which activate
RNaseH.

19. 3rd Approach :
 It combines features of both previous approaches.
 They contain both internucleotides linkages which are
responsible for RNaseH activation and which shows
resistance against them.
 Digestion of mRNA target in RNA duplex releases
oligonucleotides which are resistant against nuclease
enzyme, hence are more effective than oligonucleotides in
1st approach.
 They may form hybrids of oligodeoxyribonucleotides and
oligoribonucleotides.

20. Ribozyme
 A ribozyme is an RNA molecule, which has enzymatic activity
usually concerned with RNA degradation.
 In the ribozyme approach a DNA sequence specifying an
enzymatic RNA sequence is fused with a sequence of the
gene against which the ribozyme is aimed.
 Therefore the RNA product of this gene construct has a
sequence complementary to the sense RNA (mRNA)
produced by target gene.
 The complementary sequence of this RNA pairs with the
sense RNA produced by the target gene and the ribozyme
sequence linked to it degrades the sense RNA.

21.  It degrades the mRNA by cleaving the phosphodiester
backbone at a specific cutting site.
 Types of Ribozymes :
 RNase P
 Hammerhead Ribozyme
 Hairpin ribozyme
 Group I and group II intron splicing ribozymes.

23. Application of Antisense Technology :
1. In agriculture :
 Slow fruit softening tomato
 Changed fatty acid composition of Brassica Oil
 Delayed senescence in Carnation
 Male sterility
2. In medicine :
 Cancer Chemotherapy
 AIDS therapy
 Genetic disorders
 New drug discovery

24.  Flavr Savr Tomato :
 It is developed by Californian company Calgene.
 The genetically engineered Flavr Savr tomato was
introduced on 21 May, 1994.
 Development of Flavr Savr tomato :
 Softening of fruit is largely due to dehydration of cell wall
(pectin) by enzyme polygalacturonase (PG).
 The gene encoding PG has been isolated and cloned
(pTOM6).

25.  Procedure involves :
1. Isolation of DNA from tomato plant that encodes the enzyme
polygalacturonase (PG).
2. Transfer of PG gene to a vector bacteria and production of
complementary DNA (cDNA) .
3. Introduction of cDNA into a fresh tomato plant to produce
transgenic plant.
 Mechanism :
 In normal plants, PG gene encodes a normal or sense mRNA
that produce the enzyme PG and it is involve in fruit ripening.
 The cDNA of PG encodes for antisense mRNA, which is
complementary to sense RNA.
 The hybridization between sense and antisense mRNA
renders the sense mRNA ineffective.

26.  Consequently no polygalacturonase is produced hence fruit
ripening is delayed.

28. Role in Drug Discovery :
 In recent years, Antisense oligonucleotides (AS-OD)
technology have been widely used as potent and promising
tool for drug discovery and development.
 Diseases are connected to insufficient or excess production
of certain proteins.
 If the production of these proteins is interrupted i.e.
increased or decreased then certain diseases can be cured .
 The vast majority of drugs available today either act at the
protein level, or the drugs themselves are proteins.

29. CONCLUSION

30.  Reference :
 Plant Biotechnology – B.D. Singh
 Agricultural Biotechnology – Purohit
 www.sciencedirect.com
 www.ukessys.com