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
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.
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.
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.
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.
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.
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