Separation of Phosphodiester Oligodeoxynucleotides and Phosphorothioate Antisense Oligodeoxynucleotides by Capillary Zone Electrophoresis at a Low pH

Oligodeoxynucleotides (ODNs) possess biological activity in vivo and are used as cancer therapeutics. Synthesized ODNs contain several by-products, and therefore their purity check and resolution of single-base, i.e., the separation of ODNs differing by one nucleotide in length, become necessary. In this study, capillary zone electrophoresis (CZE) method was developed for the separation of two sets of model compounds of single-stranded oligodeoxynucleotide mixtures (18–20 mers), phosphodiester oligodeoxynucleotides (PO-ODNs), and their phosphorothioate modifications (PS-ODNs), with equal sequences differing by a single-base. The effects of the CZE operating parameters, such as the pH values and the concentrations of the running buffer, the varieties and concentrations of additives, the separation voltage as well as the temperature, on the separation were investigated and optimized to further improve the resolution. It was confirmed that the pH value of the buffer played the most important role in the separation, and the urea used as the additive in the system significantly improved the resolution of PS-ODNs. Consequently, the PO-ODNs and PS-ODNs mixtures could be single-based separated on a fused-silica capillary of 50 μm × 49.0 cm (40.7 cm of effective length) under the optimum conditions: the running buffer system of 50 mmol/L NaH2PO4-H3PO4 (pH 2.24)-7 mol/L urea, the pressure injection of 2 kPa × 10 s, the separation voltage of -20 kV, the column temperature of 25 °C, and the ultraviolet (UV) detection at 260 nm. The average resolutions for the separation of 18–19 mers and 19–20 mers of PO-ODNs were 4.68 and 3.20, respectively; the average resolutions for the separation of 18–19 mers and 19–20 mers of PS-ODNs were 1.23 and 0.81, respectively. The relative standard deviations of all the migration time and the resolution were less than 5%. This method will be useful for the quantification of PO-ODNs and PS-ODNs samples as they are used in antisense drug development because of their relatively easy operation and good reproducibility compared with the capillary gel electrophoresis.