3. INTRODUCTION
“Antisense technology is a tool that is used for the Inhibition of gene
expression. ”
Antisense therapy is a form of treatment for genetic disorders or infections.
When the genetic sequence of a particular gene is known to be causative of
a particular disease, it is possible to synthesize a strand of nucleic acid
(DNA, RNA or a chemical analogue) that will bind to the messenger
RNA (mRNA) produced by that gene and inactivate it, effectively turning
that gene "off".
This is because mRNA has to be single stranded for it to be translated.
Alternatively, the strand might be targeted to bind a splicing site on pre-
mRNA and modify the exon content of an mRNA.
4. BASIC CONCEPT
The two complementary strands of double-stranded DNA (dsDNA) are usually
differentiated as the "sense" strand and the "antisense" strand. The DNA sense
strand looks like the messenger RNA (mRNA) the DNA sense strand itself is not
used to make protein by the cell. It is the DNA antisense strand which serves as the
source for the protein code, because, with bases complementary to the DNA sense
strand, it is used as a template for the mRNA. Since transcription results in an RNA
product complementary to the DNA template strand, the mRNA is complementary
to the DNA antisense strand. The mRNA is what is used for annulation (protein
synthesis). Hence, a base triplet 3'-TAC-5' in the DNA antisense strand can be used
as a template which will result in an 5'-AUG-3' base triplet in mRNA.
5. ANTISENSE OLIGONUCLEOTIDE
The concept of antisense oligonucleotide gene silencing was first
introduced in 1978 when Stephenson and Zamecnik used an antisense
oligonucleotide to stop viral replication in cell culture.
An antisense oligonucleotide is a single strand of nucleic acid or nucleic
acid analogs, most often an oligodeoxyribonucleotide, usually 15–20
nucleotides in length with sequence complementary to a specific target
mRNA.
The antisense oligonucleotide and target mRNA bind together via Watson–
Crick base pairing, and this hybridization leads to reduced levels of
translation of the target transcript.
8. 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 particular gene.
In case of viruses, antisense
oligonucleotides inhibit viral
replication with blocking
expression of integrated
proviral genes.
Usually consist of 15–20
nucleotides.
9. 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 or 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.
10. 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.
11. 1st approach
The antisense oligonucleotides binds the target sequence causing
both “hybridisation arrest ” and “RNaseH activation”.
2nd approach
Translation arrest but they do not activate enzyme RNaseH.
Resistance against nucleuses enzyme.
eg. Oligoribonucleotides
3rd approach
It combines features of both previous approaches.
“Hybridisation arrest ” and “RNaseH activation”.
Resistance against nucleuses enzyme.
13. RNase H appears to be a ubiquitous
enzyme in eukaryotes and bacteria.
The antisense oligonucleotide
(typically a deoxyribonucleotide)
binds the target RNA to form the
heteroduplex substrate.
RNase H binds via its binding domain
at the 3′ antisense oligonucleotide/5′
RNA pole and cleaves the target RNA
approximately 7 base pairs from its
binding site.
The target mRNA is degraded,
whereas the antisense oligonucleotide
remains intact, allowing it to form
another heteroduplex substrate for
induction of RNase H cleavage.
14. 30,000
10,000
500
50
1-5
Human genome ∼
Profiling to determine
expressed genes
Profiling to determine genes
upregulated in disease
Bioinformatics
Antisense technology
Target genes
Figure : Diagramatic representation of the role of
antisense technology in drug discovery.
15. POTENTIAL ADVANTAGES
Oligonucleotides are manufactured quickly I.e.
within a week.
Sensitivity of therapy can be easily measured.
Potential to produce longer lasting responses.
Potential for enhanced binding affinity to target.
16. LIMITATIONS
Antisense agents have to be protected against
nucleolytic attack.
Large doses are required for therapeutic
response.
The difficulty in directing to a particular cells.
The half-life in plasma is short.
18. REFERENSES
http://en.wikipedia.org/wiki/Antisense_the
rapy
“Antisense oligonucleotid technologies in drug discovery” Tarek Aboul- Fadl
Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt.
“Antisense Oligonucleotide: Basic Concept and its Therapeutic Application”,
Dr Bharti Bhandari, Dr Deepti Chopra, Dr Neeta Wardhan, 1Department of
Physiology, AIIMS, Jodhpur, 2Department of Pharmacology, HIMSR,
JamiaHamdard, Department of Pharmacology, UCMS, Delhi, respectively.
Journal of Research in Pharmaceutical Science Volume 2 ~ Issue 3 (2014)
pp: 01-13
“Antisense technologies Improvement through novel chemical modifications”
Jens Kurreck Institut fu¨r Chemie-Biochemie, Freie Universita¨t Berlin,
Germany.