Oligonucleotide therapy involves using short strands of nucleic acids to alter gene expression and treat diseases. There are several techniques, including antisense oligonucleotides that bind to mRNA to inhibit translation, siRNA that induces mRNA degradation, and aptamers that bind to target proteins. Successful oligonucleotide therapy requires the oligonucleotide to be designed to target the desired gene, delivered to cells, accumulate intracellularly, and localize to active sites without compartmentalization to exert its activity. Major applications include oncology, cardiovascular and central nervous system disorders, and treating viruses, inflammation, and genetic diseases.

1. Oligonucleotide
Therapy

2. Introduction:
Oligonucleotide (ON) is a molecule composed of 25 or fewer nucleotides.
ON strategies designed to treat diseases by altering gene expression of an affected individual.
• It is complementary to a position of a gene around the site to be mutated. But it contains
mismatched for the base to be mutated.
Basic Principle:
The idea behind oligonucleotide therapy is to identify a potential target mRNA sequence, perhaps
a sequence encoding an important viral or cancer protein and to introduce the oligonucleotide to
the infected or cancerous cell so that the synthesis of the protein will be blocked.
 Existing but abnormally expressed genes are modulated
 Antisense technique- ONs are synthesized that are complementary to the RNA of interest
(control of translation)
 Antigene technique- ONs are synthesized for the direct binding to DNA (control of
transcription)

3. Technique of Oligonucleotide therapy:
 The basic technique that the starting material is a single-stranded DNA (to be mutated) carried in
an M13 phase vector
 On mixing this DNA with primer the oligonucleotide hybridizes with the complementary
sequences, except at the point of mismatched nucleotide.
 Hybridization (despite a single base mismatch) is possible by mixing at low temp with excess of
primer, and in the presence of high salt concentration
 By the addition of 4-deoxyribonucleoside triphosphates and DNA polymerases replication occurs.
Thus the oligonucleotide primer is extended to form a complementary strand of the DNA. The
ends of the newly synthesized DNA are sealed by the enzyme DNA ligase.
 The double bond stranded DNA (i.e. M13 phage molecule) containing the mismatched nucleotides
is introduced into E. coli by transformation.
 The infected E. coli cells produce M13 virus particles containing either the original wild type
sequence or the mutant sequence.
 The double-stranded DNAs of M13 are isolated. The mutated genes are cut with restriction
enzymes, ligated to a plasmid vector of E. coli. The altered protein is produced in the E. coli which
can be isolated and purified.

5. Steps in successful ON therapy
1. Design and chemistry
2. Stability
3. Cell association and entry
4. Net accumulation (influx>efflux)
5. Avoid compartmentalization
6. Localization at active sites
7. Exert activity
Delivery strategies
1. Liposomes
2. Dentrimers
3. Carrier peptide-mediated
4. Receptor mediated
5. Polymer (microsphere formulation)

6. Major class of oligonucleotides therapeutics
1) Antisense- Cleavage the target mRNA or inhibit translation by steric hindrance
2) SiRNA- Induce mRNA degradation
3) MiRNA- Inhibit translation
4) Antamer- Binds to target proteins
1) Antisense oligonucleotides
Antisense oligonucleotides are the molecules made of synthetic genetic material which interact
with the natural genetic material that codes the information for production of proteins.
An antisense oligonucleotide therapy is one such approach which blocks the proteins
formation by inhibiting translation step.
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 slicing or the translation machinery.

7. DNA mRNA
Translation
proteins
transcription
Traditional drug
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E
A
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8. Applications:
1. Oncology
2. CVS and CNS therapeutics
3. As antiviral and antibacterial agent
4. Inflammation therapeutics
2) RNA interference
RNA interference is the process of using specific sequence of double stranded RNA to
knockdown the expression level of target genes or therapeutics purpose.

9. dsRNA
cleavage
SiRNA
mRNA
SiRNA-mRNA complex formed by base pairing
Binds to
RISC (RNA induced silencing complex)
Splicing proteins

10. 3) Aptamers
Aptamers are oligonucleotides (DNA or RNA) having high affinity and specificity in
identifying the target molecules.
Aptamer- Target Binding mechanism

11. Therapeutic application of Oligonucleotide
1. Cancer therapy
2. Muscular dystrophy
3. Thalasemia
4. Arthritis
5. Diabetes
6. Asthma