PREFACE

 Pharmacy is a profession that is concerned with

the art and science of preparing and convenient
material for distribution and use in treatment and
prevention of disease, so it is fully technical
profession where practical knowledge is much more
important along with theoretical knowledge.
 According to curriculum of 4 year degree integrated
course of BACHELOR Of PHARMACY each student
has to undergo practice school for a period of 150
hours.
 I was directed a project on “Antisense Technology”
and this report contains a brief description of the
above topic which was observed during the program.
INSTITUTE OF PHARMACEUTICAL SCIENCES
KURUKSHETRA UNIVERSITY,
KURUKSHETRA
DECLARATION

I hereby declare that project work embodied in the

entitled , carried out by me under the supervision of
Dr. Sukhbir Lal Khokar , Professor,
Kurukshetra University,
Kurukshetra

I am indebted to my institutional guide for their step

to step guidance throughout the preparation of
project report.
 Introduction
Diseases are often occurred in our body when there is
more production of protein then needed or there is lack
of production of protein, due to which various diseases
or disorders starts occurring in our body.1
If the production of protein is controlled externally by
using of various therapies or drugs then many diseases
can be treated or cured. There are lots of disorders like
cancer, viral, parasitic infections which cause to
production of a lot of protein in our body.1
The production of protein can be inhibited by blocking
the steps of transcription or translation. The production
of protein can be controlled by blocking transcription
step and is called antigene therapy, while when the
translation step is blocked of protein formation then it is
called antisense therapy or antisense technology.1
It is the tool that is used for the inhibition of gene
expression. The main mechanism behind it is that when
an antisense nucleic acid pairs it with the
complementary RNA molecule and prevents its
translation step due to which protein production is
inhibited. In antisense technology technique, short
segments of single stranded DNA called
oligodeoxynucleotide are introduced.4

These short segments of single stranded DNA that is

oligonucleotides are complimentary to the mRNA.
These technologies have become very powerful weapon
for studying gene function and for discovering more
specific disease treatment.1
 Protein Formation

Protein is formed in our body and protein is regularly

produced in our body. The protein formation occurs
in two steps 2
Step 1 Transcription
Step 2 Translation

(Transcription)
Transcription: - It is a process in which RNA copy i.e
ribonucleic acid is formed from genes of DNA sequence.
The copy formed is messenger RNA (mRNA) and it carries
protein information which is encoded in DNA.3
Translation:- It is a process in which ribosomes or
endoplasmic reticulum in the cytoplasm synthesize protein
which was done after the transcription step.
The whole process that is transcription and translation is
known as gene expression.
In translation, the mRNA was decoded in ribosome to form
specific amino acid chains.3

Translation step is carried out in three steps :-

1. Initiation: The organelle assembles round the target
ribonucleic acid. The primary RNA is connected at the
beginning sequence.
2. Elongation: The last RNA valid by the little
ribosomal fractional monetary unit (accommodation)
transfers the amino acid it carries to the big ribosomal
fractional monetary unit that binds it to the one
amongst the preceding admitted RNA
(transpeptidation). The organelle then moves to
ensuing ribonucleic acid sequence to continue the
method, making amino acid chain.
3. Termination: Once a stop sequence is reached, the
organelle releases the peptide. The ribosomal
advanced remains intact and moves on to ensuing
ribonucleic acid to be translated.
 The Basics Of Antisense

 Sense refers to the original sequence of DNA or RNA

while Antisense refers to complementary sequence.1
 When complementary molecules bind to the mRNA,
it blocks the protein production.1
 mRNA i.e messenger RNA acts like a carrier which
carries genetic information from DNA to other cells
which are involved in protein production.1
 When antisense drugs bind with the mRNA, it
inhibits the specific disease related proteins.1
 A sense strand of DNA molecule is 5’ to 3’ mRNA
molecule.4
 In this technology, when an antisense strand or
complementary sequence of DNA binds to the
mRNA, a cell will detect the double helix as foreign
matter to the cell and will start proceeding to degrade
the faculty mRNA molecule which prevents the
production of useless or undesired protein.1
 A DNA antisense molecule must be approximately
17 bases while RNA have 13 bases.
 RNA antisense strands can be of two types:-
  Catalytic
 Non-catalytic.
 Catalytic antisense which also known as ribozymes
will cut the RNA molecule at specific sequences.
 Non catalytic RNA antisense strands will block the
RNA processing.
 The exact mechanism of antisense strand is not
known while the current hypothesis include
“Blocking RNA splicing, accelerating degradation of
the RNA molecule, and preventing introns from
being sliced out of the mRNA, impending the
exportation of mRNA into the cytoplasm, hindering
translation and the triplex formation in DNA.1
 Antisense Oligonucleotides
 The effect of antisense in oligonucleotides sequence was
first demonstrated by Stephenson and Zamecnik in 1970, in
rous sarcoma virus.
 Antisense oligonucleotides consist of 15-20 nucleotides,
which are complementary to their target mRNA.5
 When these oligonucleotides combines with the target
mRNA, a DNA or RNA hybrid form , which is degraded
by the enzyme RNase H.
 RNase H is non-specific endonuclease, which is catalyses
the cleavage of RNA.5

Types of Antisense Oligonucleotide5

1. First generation AS-ON
2. Second generation AS-ON
3. Third generation AS-ON
First generation AS-ON
 It was synthesized by Eckstein.
 In this Phosphorothioate which is oligo deoxy
nucleotide is the major representative of First
generation DNA analogs.
 In first generation AS-ON, Phosphorothioate linkages
in Oligonucleotide is used to enhance the nuclear
resistance.
 They were first used for the inhibition of HIV.
 They have ability to activate RNase H.

Second generation AS-ON

 In this generation it contains nucleotides with alkyl
modifications which was at the 2’ position of the
ribose.
 They have high affinity to target mRNA.
 They are less toxic than first generation AS-ON.
 The most important members of this generation are 2’-
O-methylethyl RNA and 2’-O-methoxy ethyl RNA.
Third generation AS-ON
 They are most newest and most promising.
 They enhance the binding affinity and biostability.

Some examples are

 Peptide nucleic acids (PNAs)
 Locked nucleic acid (LNA)
 Tricyclo-DNA (tcDNA)
 Cyclohexene nucleic acids (CeNA)
Working of Antisense Technology
The main objective of antisense technology is to block
the production of protein which is causing disease. This
is done by formation of synthetic antisense or
complementary nucleotide sequence which interacts
and binds with the original mRNA sequence.
The complex formed of mRNA and antisense was no
longer be translated and the protein which are causing
disease cannot be produced, hence the disease is treated.
1
 Steps of working

 The main objective of antisense technology is to

block the production of proteins which is causing
disease.2
 A complimentary DNA sequence is cloned.
 The cloned DNA sequence was inserted in front of
the sequence. It means that the last base of the
inserted DNA is complimentary to first base of the
original DNA.
 The second last base is complimentary to the
second base and so on.
 When transcription takes place in the modified
strand of DNA, the mRNA becomes double
stranded. 1
 Depending on the size of the inserted DNA, the
mRNA could be double stranded or could be
partially double stranded.
 As a result of the double strand mRNA, ribosome
finds it difficult to process mRNA and very little
protein is produced and the disease can be treated.1
 Applications

Antisense technology is used in the treatment of various

disease like
 Cancer
 Viral Infections
 Cardiovascular Disease
 Neurological Disease
 Immunological Disease
 Ocular Diseases1
 Invitro application: Antisense Technology is very
useful and is applied in researches in understanding the
function of genes. It is useful in studying the knock on
effects of translation step in protein production which
can determine the cellular process in which the protein
is involved in.6
 Antisense Technology plays a crucial role in producing
high yielding, disease and pest resistant, good
nutritional value crop varieties. 6
 Antisense oligonucleotides are artificially produced
which is used in the treatment of disorders which are
involving gene mutation.6
 Bibliography

1. Stuti Gupta, Ravindra Pal Singh, Nirav Rabadia,

Gaurang Patel, Hiten Panchal, Review Article,
Antisense Technology
https://globalresearchonline.net/journalcontents/
volume9issue2/article-007.pdf
2. U. Satyanarayan, U. Chakrapani, Biochemistry,
Fourth edition 2013, Published by Elsevier.
3. Dr U. Satyanarayan, Biotechnology, 2010, Books
And Allied Pvt Ltd.
4. Bochet, Convreur , Fattal, Intravitreal administration
of antisense oligonucleotides , 2000, (19),
131-147.
5. Antisense Technology, Desh Bandhu Gangwar,
M.Tech Biotech, Dr. Gunjan Garg, Assistant
Professor, School of Biotechnology.
https://www.slideshare.net/DeshBandhuGangwar/
antisense-technology

6. https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC3117510/
THANK
YOU