Oligonucleotides are short strands of DNA or RNA that are synthesized using solid-phase chemical synthesis. They are made by attaching nucleotide monomers individually and sequentially to a solid support such as controlled pore glass or polystyrene beads. The phosphoramidite method is commonly used, involving cycles of deprotection, coupling of the next nucleotide, oxidation to stabilize the linkage, and capping. Solid phase synthesis allows for automation and purification after each step. However, oligonucleotides face challenges including degradation by nucleases and short plasma half-life that limit their therapeutic potential.

1. Oligo-nucleotideSynthesis:
Presentation By:
Tahira Jabeen
Iqra Ramzan

2. Oligonucleotides
 Oligonucleotides are short, single-stranded DNA or RNA
molecules that have a wide range of applications in genetic
testing, research, and forensics.

3. Oligonucleotide
sSynthesis
 Oligonucleotides are made in the laboratory by solid-phase
chemical synthesis, these small bits of nucleic acids can be
manufactured with any user-specified sequence, and so are vital
for artificial gene synthesis, polymerase chain reaction (PCR),DNA
sequencing, library construction and as molecular probes.

4. SolidSupport
forOligo
Synthesis
 The oligonucleotides are synthesized on solid supports from the
3’-end and the first monomer at this end is normally attached to a
CPG(Controlled Pore Glass) or Polystyrene (PS).

5. SolidSupport
forOligo
Synthesis
Controlled
PoreGlass
(CPG) :
 Controlled-pore glass is rigid and non-swelling with deep pores in
which Oligonucleotides synthesis takes place.Glass supports with
500 Å (50 nm) pores are mechanically robust and are used
routinely in the synthesis of short Oligonucleotides. However,
synthesis yields fall off dramatically when Oligonucleotides more
than 40 bases in length are prepared on resins of 500 Å pore size.
This is because the growingOligonucleotides blocks the pores and
reduces diffusion of the reagents through the matrix. Although
large-pore resins are more fragile, 1000 Å CPG resin has proved to
be satisfactory for the synthesis of oligonucleotides up to 100
bases in length, and 2000Å supports can be used for longer
oligonucleotides.

6. SolidSupport
forOligo
Synthesis
Polystyrene
(PS) :
 Highly cross-linked polystyrene beads have the advantage of good
moisture exclusion properties and they allow very efficient
oligonucleotide synthesis, particularly on small scale (e.g. 40
nmol).

7. Oligonucleotid
eSynthesis
Method
Phosphoramid
iteSynthetic
Method
 McBride and Caruthers in 1983, developed this method of
oligonucleotide synthesis.

8. Processes:
De-protection
Coupling
Capping.
1
2
3

9. 1
Step
De-protection:
In the classic de-protection step the trityl group attached to
the 5’ carbon of the pentose sugar of the recipient nucleotide
is removed by trichloroacetic acid (TCA) leaving a reactive
hydroxyl group.

10. 2
Step
Coupling:
In the coupling step, the Phosphoramidite monomer is added in the
presence of an activator such as a tetrazole, a weak acid that attacks the
coupling phosphoramidite nucleoside forming a tetrazolyl phosphoramidite
intermediate.This structure then reacts with the hydroxyl group of the
recipient and the 5’ to 3’ linkage is formed .The tetrazole is reconstituted
and the process continues.

11. 3
Step
Oxidation:
The oxidation step stabilizes the phosphate linkage in the
growing oligonucleotide.The traditional method of achieving
this is by treatment with iodine in water.

12. 4
Step
Capping:
The final step of the synthesis cycle is the capping reaction. Any remaining
free 5’hydroxyl groups are blocked at the capping step in an irreversible
process.This step prevents the synthesis of oligonucleotides with missing
bases. Following this step, the oligonucleotide is ready for the next
monomer.

13. Advantages of
Solid Phase
Synthesis:
 Large excesses of solution-phase reagents can be used to drive
reactions quickly to completion
 Impurities and excess reagents are washed away and no
purification is required after each step
 The process is amenable to automation on computer-controlled
solid-phase synthesizers.

14. Disadvantages
or limitations:
 Antisense agents have to be protected against nucleolytic attack.
The pharmacokinetic characteristics of oligonucleotide drugs is
similar to that of naturally occurring nucleic acids, makes them
poor therapeutic candidates, as they are immediately degraded in
vivo by nucleases and do not have adequate pharmacokinetic
properties.
 Larger dose are required for therapeutic response.
 The difficulty in directing to a particular cells.
 The half-life in plasma is short. Department of Pharmacy,

15. Problems and
Challenges:
 Monitoring coupling efficiency is critical parameter to get high
yield of Oligo synthesis. If the coupling efficiency is 99% then,
theoretical yield for a 24mer will be 89.1% full length product
(FLP) at 99.5% average coupling efficiency and 79.4% FLP at
99.0% average coupling efficiency.
 Even a 0.5% average coupling failure rate can be dramatic for
longer oligonucleotides.
 A minor increases in average coupling failure rates will have a
substantial net effect on even average length oligonucleotides. It
is for this real-time monitoring of every custom synthesis reaction
on every synthesis platform.