Oligonucleotides therapeutics work by altering gene expression through complementary binding to mRNA. There are three major classes: antisense oligonucleotides inhibit protein expression through hybridization to mRNA, siRNA induce mRNA degradation, and miRNA inhibit translation. Oligonucleotides are modified through conjugation or encapsulation to improve delivery and stability, and resist nuclease degradation. Antisense oligonucleotides specifically designed to hybridize with mRNA can function through RNase H degradation or steric blocking of translation.

1. Oligonucleotides therapeutic

2. Definition
Oligonucleotide (ON) is a molecule composed of 25 or fewer
nucleotides.
ON strategies designed to treat disease by altering gene
expression of an affected individual.

3. Major class of oligonucleotides therapeutics
Class General M.O.A Specific M.O.A
1. Antisense Inhibit protein cleavage the target mRNA
expression via or inhibit translation by
complementary hybridiz- steric hindrance
ation to mRNA
2.SiRNA induce mRNA degradation
3.miRNA Inhibit translation

4. Characteristics
1. The majority of Ots are chemically modified to block nuclease based
metabolism and prolong duration of action.
2. Many Ots are chemically conjugated or encapsulated to facilitate delivery ,
block renal filtration.
e.g; cholesterol , lipid nanoparticles , small molecules functional gp etc.
3. Ots are highly “tunable” by site directed chemical modification, may of
which are now well characterized.

5. Chemical Modifications

7.  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 .
Antisense Oligonucleotides

8.  On the basis of mechanism of action
Two classes of antisense oligonucleotides can be discerned
1.The Rnase H-dependent oligonucleotides ,which induce the
degradation of mRNA
2. The Steric-blocker oligonucleotides, which physically prevent or
inhibit the progression of splicing or the translational machinery.

10. Antisense oligonucleotide design
The strength & stability of interaction between the ASO & complementry target
mRNA depend on factors such as thermodynamic stability.
 We need to consider at least four parameter in ASO design in order to increases
the hit rate.
1. Prediction of secondary structure of RNA.
2. Identification of preferable RNA secondary local structures.
3. Motifs searching & GC content calculation,
4. Binding energy prediction.

11. Chemical modification of ASo
Various chemical modification have been developed o enhance nuclease resistance, prolong tissue half-
life, increases affinity & potency & reduce non-sequence specific toxicity.
•Three types of generation as follows:
1. First generation ASO ; Phosphoro-thioate-deoxy-nucleotides are the first nucleotides & have a
sulfur atom replacing the non-binding backbone.
2. Second generation ASO ; containing nucleotides with alkyl modification at the 2’position of the
ribose
3. Third generation ASO ; newest & most promising .
enhance binding affinity & biostability.
Chemical modification of the furanose ring of the nucleotide.

12. Third generation ASO
Newest and most promising.
Enhance binding affinity and biostability.
Peptide nucleic acids (PNAs)
Locked nucleic acid (LNA)
Tricyclo-DNA (tcDNA)
Cyclohexene nucleic acids (CeNA)

14. In PNAs the deoxyribose phosphate backbone is
replaced by polyamide linkages.
The property of high-affinity nucleic acid
binding can be explained by the lack of
electrostatic repulsion because of the absence of
negative charges on the PNA oligomers.
The antisense mechanism of PNAs depends on
steric hindrance.
PNA

15. The ribose ring is connected by a methylene bridge (orange)
between the 2’-O and 4’-C atoms thus “locking” the ribose ring in
the ideal conformation for Watson-Crick binding.
Thus the Pairing with a complementary nucleotide strand is
more rapid and increases the stability of the resulting duplex
LN
A

18. Aptamers special features
oAptamer have similar affinites as antibiocts to the target
molecule.
oGreater stability.
oLow Immunogenicity.
oEasier large scale production.

19. RNA Interference
RNA interferenc is the process of using specific sequence of double stranded
RNA to knockdown the expression level of taget genes or therapeutic purpose.
dsRNA
cleavage
SiRNA
mRNA
SiRNA-mRNA complex formed by base pairing
bind to
RISC (RNA induced silencing complex)
splicing protein

21. References
www.niper.ac.innucleic.pdf
•Nus.edu.sgngsstudent achieve papers2006 antisense
oligonucleotidedesigntherapeuticapplication.pdf
•https:en.wikipedia.orgwikioligonucleotide
•https:www.ncbi.nlm.nih.govpubmed25093706

22. Delivery vectors can take care of both toxicity and drug delivery
problems .The vector can also protect the drug from degradation
and also from rapid clearance from the body. The vector must:
Be of small size to allow intercalation between tissues.
To allow intracellular transport, they must be non-toxic and stable
in the blood stream
Delivery vectors:

23. They must retain the drug when in the circulation, and
Must release it at its target before elimination.
These are quite challenging tasks but many ideas have been
developed such as liposomes, protein or peptide constructs
and polymers.

24. Application of Antisense Oligonucleotides:
Antisense drugs are being researched to treat a variety of
diseases such as :
Lung cancer,
Colorectal carcinoma
Pancreatic carcinoma
Malignant melanoma
Diabetes
Amyotrophic lateral sclerosis (ALS),
Asthma,
Arthritis.