Manufacture of oligos

In contrast to the biopharmaceuticals discussed thus far (recombinant proteins and gene therapy products), antisense oligonucleotides are manufactured by direct chemical synthesis. Organic synthetic pathways have been developed, optimized and commercialized for some time, as oligonu-cleotides are widely used reagents in molecular biology. They are required as primers, probes and for the purposes of site-directed mutagenesis.

The nucleotides required (themselves either modified or unmodified as desired) are first reacted with a protecting chemical group. Each protected nucleotide is then coupled in turn to the growing end of the nucleotide chain, itself attached to a solid phase. After coupling, the original protecting group is removed and, when chain synthesis is complete, the bond anchoring the chemical to the solid phase is hydrolysed, releasing the free oligo. This may then be purified by HPLC. The most common synthetic method used is known as the phosphoramidite method, which uses a dimeth-oxytrityl protecting group and tetrazole as the coupling agent. Automated synthesizers that can quickly and inexpensively synthesize oligos of over 100 nucleotides are commercially available.

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