Oligonucleotides are typically made in the laboratory using phosphoramidite chemistry and solid-phase synthesis. This method begins by attaching a starting nucleoside to a solid support that is typically composed of polystyrene (PS) or controlled pore glass (CPG). Then the following protocol is used to synthesize oligonucleotides:
Step 1. Detritylation - Before any additional nucleosides can be added, the 5'-4,4'-dimethoxytrityl (5'-DMT) protecting group must first be removed from the support-bound nucleoside.
Step 2. Activation & Coupling – After detritylation the support-bound nucleoside is now primed to react with the next base in the oligo sequence, this is added as a nucleoside phosphoramidite monomer. The nucleoside phosphoramidite is dissolved in acetonitrile (ACN) and mixed with an activator (e.g. tetrazole) so that it can react with the 5'-hydoxyl group of the support-bound nucleoside.
Step 3. Capping – Any support-bound nucleosides with unreacted 5'-hydroxyl groups must be capped (i.e. blocked) or they will react during the next cycle resulting in an oligonucleotide with a missing base. Unreacted 5'-hydroxyl groups can be blocked using a mixture of two capping solutions consisting of acetic anhydride and N-methylimidazole (NMI).
Step 4. Oxidation – The bond of the newly attached nucleoside phosphoramidite is then stabilized via iodine oxidation.
Step 5. Detritylation – The 5'-DMT protecting groups is removed from the support-bound nucleoside chain. This allows the 5'-hydroxyl group to react with the next nucleoside phosphoramidite monomer. This cycle is repeated, once for every base to be added in the sequence, until the oligonucleotide is complete.
Step 6. Cleavage – The finished oligonucleotide is removed or cleaved from the solid support.
Step 7. Deprotection – Following cleavage, the oligonucleotide, which is dissolved in concentrated aqueous ammonia, is heated to remove protecting groups from the bases and phosphates.