The solid phase synthesis of oligonucleotide using Phosphoramidite methods is the most common (chemical synthetic) method.  It is highly efficient, fast coupling with stable start materials. Phosphoramidite method is carried out in the solid material, where an oligonucleotide being added is covalently bound, via its 3’-terminal hydroxy group. Chemical oligonucleotide synthesis is carried out from 3’ to 5’ direction.  The adjacent nucleotides are connected through 3’ to 5’ phosphate diester internucleosidic linkage.

Synthesis steps:

Deblocking (detritylation)

An oligonucleotide’s protection group of DMT, being pre-attached to CPG, is removed by a solution of an acid, such as trichloroacetic acid, resulting in a free 5’ hydroxyl group on the first base.

Coupling

A nucleotide phosphoramidite, the building block of DNA synthesizing, is activated by an acidic azole catalyst, tetrazole on its 3’ terminate.  The 5’ terminate hydroxy group is still protected by DMT. This mixture is brought in contact with the starting solid support (first coupling) or oligonucleotide precursor (following couplings) whose 5’-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage.

Capping

After the completion of the coupling reaction, a small percentage of the solid support-bound 5’-OH groups (less than 2%) remain unreacted and need to be permanently blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion commonly referred to as (n-1) short mers.This is done by acetylation of the unreacted 5’-hydroxy groups using a mixture of acetic anhydride and 1-methylimidazole as a catalyst.

Oxidation

The newly formed tricoordinated phosphite triester linkage is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine) oxidizes the phosphite triester into a tetracoordinated phosphate triester.

Through above synthetic steps, an oligonucleotide is coupled to solid support material. Carried out by a stepwise synthetic cycle, additional nucleotides are coupled until all of the desired bases are added.

During the reaction, the synthetic efficiency can be determined by observing the solution color from TCA treatment.

After synthesis is complete, the fully protected, solid support-bound oligonucleotides are subjected to deprotection. Most often, 5’-DMT group is removed at the end of the automatic synthesis. The oligonucleotides are then released from the solid phase and deprotected (base and phosphate) by treatment with aqueous ammonium hydroxide.

Purification

The fully deprotected product is used as is, or the desired oligonucleotide can be purified by a number of methods. Most commonly, the crude product is desalted using C18 column. To eliminate unwanted truncation products, the oligonucleotides can be purified via polyacrylamide electrophoresis followed by desalting.  The yields are determined by OD260.  The products are vacuumed dried and packed according to the requirement.