This document discusses antisense oligonucleotides, which are short DNA or RNA strands that bind to messenger RNA to inhibit gene expression. It describes the mechanism of action, different generations of oligonucleotides including modifications to improve stability and binding, and applications for disease treatment. First generation oligonucleotides used phosphorothioate modifications and activated RNase H cleavage of mRNA. Second generation used 2' modifications like methyl and prevented RNase H cleavage. Newer generations like PNA, LNA, and CeNA have increased binding and stability. Antisense oligonucleotides have been approved to treat conditions like CMV retinitis and familial hypercholesterolemia.

1. ANTISENSE OLIGONUCLEOTIDES
Presented By:
Mohd Ashhar Suhail Nomani
M. Pharm 2ND Sem
Department of Pharmacology
Jamia Hamdaed, New Delhi 1

2. CONTENTS
• Introduction
• Mechanism of action of antisense oligonucleotides
• Classes of oligonucleotides
• First generation Antisense oligonucleotides
• Second generation Antisense oligonucleotides
• Third generation Antisense oligonucleotides
• Applications
• Limitations
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3. INTRODUCTION
• Antisense Oligonucleotides are unmodified or
chemically modified ssDNA, RNA or their analogs.
• They are 13-25 nucleotides long and are specifically
designed to hybridize to the corresponding mRNA by
Watson-Crick binding.
• In this technique short segments of single stranded
DNA called Oligodeoxynucleotides are introduced
in to the cell.
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4. • These Oligonucleotides are complementary to mRNA and
physically bind to it.
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5. • The antisense effect of oligonucleotide sequence
waw first demonstrated in 1970s by Zamecnik and
Stephenson, in Rous sarcoma virus.
• When these oligonucleotides combined with target
mRNA, a DNA/RNA hybrid is formed, which is
degraded by enzyme Rnase H.
• RNaseH is a non specific endonuclease, catalyzed the
cleavage RNA via hydrolytic mechanism.
• It cleaves 3’-O-P bond of RNA in DNA/RNA duplex.
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6. MECHANISM OF ACTION OF ANTISENSE
OLIGONUCLEOTIDES
• In this technique, short segments ssRNA
are introduced.
• These oligonucleotides are complement
-ary to mRNA, which bind to mRNA.
• So, they block the expression of particul
-ar gene.
• In case of viruses, antisense oligonucleotides
inhibit viral replication with blocking
expression of integrated proviral genes. 6

7. • Despite the simplicity of the idea behind Antisense
oligonucleotides, several problems have to be overcome for
successful application:
• Accessible sites of target RNA for oligonucleotide binding
have to be identified.
• Antisense agents have to protected against nuclease enzyme
attack.
• Cellular uptake and correct intracellular localization.
• It is therefore necessary to chemically modify antisense
oligonucleotides to make them stable in cells.
• Modification of phosphodiester backbone is likely to inhibit
nuclease action and several phosphodiester backbone
analogues have been developed with thios goal in mind.
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8. CLASSES OF OLIGONUCLEOTIDES
• On the basis of mechanism of action, two classes of
antisense oligonucleotides can be identified:
• The Rnase-H-dependent oligonucleotides, which
induce the degradation of mRNA
• The steric-blocker oligonucleotides, which
physically prevent or inhibit progression of splicing or
translational machinery.
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9. FIRST GENERATION ANTISENSE
OLIGONUCLEOTIDES
• First synthesised by Eckstein and collagues in 1960s.
• Phosphoro-thioate-deoxy-nucleotide are the first gen.
oligonucleotides and have sulfur atom replacing the
non bridging oxygen of sugar phosphate backbone. It
preserves the overall charge and can also activate
RNaseH for degradation of mRNA.
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10. CHARECTERISTICS OF FIRST GEN.
• Better stability to nucleases but still degrades.
• Can activate Rnase H.
• Are highly soluble and have excellent antisense
activity.
• They were first used as antisense oligonucleotides for
inhibition og HIV.
• Cannot cross lipid bilayer because of their charge and
polarity.
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11. SIDE EFFECTS
• Thrombocytopenia
• Fever
• Fatigue
• Rashes
• Luekopenia
• There is also transient inhibition of clotting times
shown by an increased activated partial
thromboplastin time(aPTT)
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12. SECOND GENERATION ANTISENSE
OLIGONUCLEOTIDES
• Second generation Antisense oligonucleotides
containing nucleotides with alkyl modifications at 2’
position of ribose
• 2’-O-methyl and 2’-O-methoxy-ethyl RNA are most
important member of this class.
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13. 13
• These second gen. oligonucleotides are resistant to
degradation by cellular nucleases and hybridize
specifically to their target mRNA with higher affinity
than phosphodiester or phosphorothioate.
• However such antisense effects result from Rnase H
independent mechanisms.

14. CHARECTERISTICS OF SECOND GEN.
• Mechanism of action for 2’ modified oligonucleotides
do not rely on Rnase H activation but on translation
arrest by blocking 80s ribosome complex formation
as well as with splicing interference.
• They were developed to try and avoid toxicity
associated with first generation AS-ONs.
• Show high binding affinity to target mRNA.
• Best stability to nucleases.
• Less toxic than first gen. AS-ONs.
• Higher lipophilicity compared to first gen.
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15. THIRD GENERATION ANTISENSE
OLIGONUCLEOTIDES
• Newest and most promising.
• Enhanced binding affinity and biostability.
• Peptide nucleic acids(PNAs)
• Locked nucleic acids(LNA)
• Tricyclo-DNA
• Cyclohexene nucleic acids(CeNA)
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16. PEPTIDE NUCLEIC ACIDS(PNA)
• In PNAs the deoxyribose phosphate backbone is
replaced by polyamide linkages.
• The property of high affinity nucleic acid binding
can be explained by lack of electrostatic repulsion
because of absence of negative charges on PNA
oligomers.
• The antisense mechanism of PNAs depends on
steric hindrance.
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17. LOCKED NUCLEIC ACIDS(LNA)
• The ribose ring is connected by methylene
bridge between 2’-O and 4’-C atoms thus
“locking the ribose ring”
• Thus pairing with complementary
nucleotide strand is more rapid and
increases stability of resulting duplex.
• LNA oligonucleotides exhibit
unprecedented thermal stability when
hybridized to a complementary DNA/RNA
strand.
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18. CYCLOHEXENE NUCLEIC ACIDS(CENA)
• The replacement of furanose moiety of DNA by a
cyclohe.xene ring gives Cyclohexene nucleic acids.
• CeNA is stable against degradation in serum and a
CeNA/RNA hybrid is able to activate Rnase H,
resulting in cleavage of RNA strand.
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• These chemical modifications change the properties of
natural oligonucleotides in following way:
• Increase RNA affinity.
• Increased hydrophobicity.
• Increased stability towards nucleolytic degradation
• Inability to elicit Rnase H activity.

20. APPLICATIONS
• Antisense oligonucleotide therapy
• Oncology
• CNS and CVS therapeutics
• As antiviral and antibacterial agent e.g. Fomivirsen
• Inflammation therapeutics
• Diabetes
• Amyotrophic lateral sclerosis(ALS)
• Asthma
• Arthritis
• Duchene muscular dystrophy
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21. LIMITATIONS
• Large doses are required for therapeutic response
• The difficulty in directing to particular cells
• Half life in plasma is short
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22. OLIGONUCLEOTIDE DRUGS APPROVED
BY US-FDA
• Fomivirsen (1998)- marketed as Vitravene for
treatment of cytomegalovirus retinitis.
• Mipomersen (2013)- marketed as Kynamro for
treastment of homozygous familial
hypercholesterolemia.
• Eleplinsen (2016)- marketed as ExondlysSI for
Duchene Muscular Dystrophy.
• Nolersen (2018)- marketed as Tegsedi for
Amyloidosis and Polynueropathy.
• Casimersen (2021)- marketed as Amondys 45 for
Duchenne Muscular Dystrophy.
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23. REFERENCES
• Genetic Engineering by Smita Rastogi and Neelam
Pathak.
• Antisense Oligonucleotides Technology in drug
discovery(DOI: 10.1517/17460441.1.4.285)
• Antisense Drug Technology Principlea stragies and
Applications by Stanley , T.Crooke
• https://www.slideshare.net/ Antisense drugs and
Oligonucleotides by Dr. Mohit Kulmi
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