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Updated: Jul 15, 2026
Chemical Triphosphorylation of Oligonucleotides
Published on: June 2, 2022
[Separation of phosphodiester oligodeoxynucleotides and phosphorothioate antisense oligodeoxynucleotides by capillary
Qian Li1, Rong Chen, Yuqing Sun
1Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 210038, China. lqianlqian@yahoo.com.cn
Abstract:
Oligodeoxynucleotides (ODNs) may possess biological activity in vivo, and are used for the cancer therapeusis. Synthesized ODNs contains many by-products, and so 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 in a single base. The effects of the CZE operating parameters on the separation were investigated and optimized to further improve the resolution, such as the pH values and the concentrations of running buffer, the varieties and concentrations of additives, the separation voltage as well as the temperature. 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 improved significantly 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 microm x 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 x 10 s, the separation voltage of -20 kV, the column temperature of 25 degrees 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; and 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 the migration time and the resolution were all less than 5%. This method will be useful for the qualification of PO-ODNs and PS-ODNs samples as they are used in antisense drug development due to the relatively easy operation and good reproducibility of the method in comparing with the capillary gel electrophoresis.
Insights
A new capillary zone electrophoresis (CZE) method effectively separates single-base differences in synthesized oligodeoxynucleotides (ODNs), crucial for cancer therapeutics. This technique ensures purity for phosphodiester (PO-ODNs) and phosphorothioate (PS-ODNs) modifications, vital for drug development.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Molecular Biology
Context:
- Oligodeoxynucleotides (ODNs) are increasingly used in cancer therapeutics due to their in vivo biological activity.
- Synthesized ODNs often contain by-products, necessitating rigorous purity checks and single-base resolution.
- Distinguishing ODNs that differ by only one nucleotide is critical for quality control and therapeutic efficacy.
Purpose:
- To develop and optimize a capillary zone electrophoresis (CZE) method for the precise separation of single-stranded oligodeoxynucleotide (ODN) mixtures.
- To investigate the impact of various CZE operating parameters, including buffer pH, additives (urea), voltage, and temperature, on separation resolution.
- To achieve single-base resolution for both phosphodiester oligodeoxynucleotides (PO-ODNs) and their phosphorothioate modifications (PS-ODNs).
Summary:
- A capillary zone electrophoresis (CZE) method was optimized for separating ODNs differing by a single base.
- The study identified pH and urea concentration as key factors for improving resolution, particularly for PS-ODNs.
- Optimal conditions achieved single-base separation for 18-20 mer PO-ODNs and PS-ODNs with good reproducibility.
Impact:
- Provides a robust and reproducible analytical method for the quality control of ODN-based therapeutics.
- Facilitates the accurate qualification of PO-ODNs and PS-ODNs used in antisense drug development.
- Offers an alternative to capillary gel electrophoresis with easier operation and comparable performance for ODN purity assessment.
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