Phosphorothioate oligonucleotides

Phosphorothioate oligonucleotides are used in antisense biotechnology as regulators of gene expression. Compared with unmodified oligonucleotides, they have increased resistance to cellular nucleases.

They are synthesized on an automated synthesizer by the phosphoramidite method using a sulfur-containing oxidizing reagent at the oxidation stage. The method allows a phosphorothioate linkage to be introduced at any position in the oligonucleotide chain. Synthesis produces 2ⁿ diastereomers, where n is the number of phosphorothioate linkages. HPLC and gel electrophoresis used for oligonucleotide purification do not separate these isomers.

After synthesis, the oligonucleotide is cleaved from the polymer support, deprotected and purified by reverse-phase HPLC or polyacrylamide gel electrophoresis.

Physicochemical properties

The isomers have different hybridization properties. Because sulfur has a higher molecular weight than oxygen, phosphorothioate oligonucleotides migrate more slowly in polyacrylamide gel. They are more hydrophobic and are retained on a chromatographic column longer than unmodified oligonucleotides. They are poorly phosphorylated by polynucleotide kinase (5–10% ³²P-labeled product) and form less stable duplexes with RNA and DNA than their unmodified analogues.