Oligonucleotide Synthesis – Phosphoramidite Synthetic Method
McBride and Caruthers in 1983, developed this method of oligonucleotide synthesis.Custom oligonucleotide synthesis begins with specification of the desired sequence in an oligonucleotide synthesis platform. Specification is composed of three crucial elements: the actual sequence that is to be made, the identification of any desired modifications, and verification of the scale at which the synthesis is to be carried out. This third element determines the choice of a column in which the synthesis will be performed. Synthesis columns
Controlled-pore glass is rigid and non-swelling with deep pores in which oligonucleotide 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 growing oligonucleotide 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 2000 Å supports can be used for longer oligonucleotides.
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).
Solid supports for conventional oligonucleotide synthesis are typically manufactured with a loading of 20-30 μmol of nucleoside per gram of resin. Oligonucleotide synthesis at higher loadings becomes less efficient owing to the steric hindrance between adjacent DNA chains attached to the resin; however, polystyrene supports with loadings of up to 350 μmol / g are used in some applications, particularly for short oligonucleotides, and enable the synthesis of large quantities of oligonucleotides.
The Oligonucleotide Synthetic Cycle
- De-protection,
- Coupling,
- Oxidation and
- Capping.
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.
Oxidation Step – Oligonucleotide Synthesis:
The oxidation step stabilizes the phosphate linkage in the growing oligonucleotide. The traditional method of
achieving this is by treatment with iodine in water.
Advantages of Solid Phase Synthesis
Solid-phase synthesis is widely used in peptide synthesis, oligonucleotide synthesis, oligosaccharide synthesis and combinatorial chemistry. Solid-phase chemical synthesis was invented in the 1960s by Bruce Merrifield, and was of such importance that he was awarded the Nobel Prize for Chemistry in 1984.
Solid-phase synthesis is carried out on a solid support held between filters, in columns that enable all reagents and solvents to pass through freely. Solid-phase synthesis has a number of advantages over solution 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.
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.
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