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Updated: Apr 6, 2026

Chemical Triphosphorylation of Oligonucleotides
Published on: June 2, 2022
Systematic optimization of gel-free capillary zone electrophoresis for phosphodiester oligonucleotide separations at
Jakob Haglöf1, Julia Regnell1, Cari E Sänger-van de Griend2
1Department of Medicinal Chemistry, Uppsala University, Box 574, Uppsala, SE-751 23, Sweden.
Background:
Reliable analysis of oligonucleotides is essential for therapeutic development and biomarker studies, yet conventional methods often rely on gels or high-salt buffers that hinder mass spectrometry compatibility. MicroDNA and microRNA are commonly analyzed using liquid chromatography or capillary gel electrophoresis (CGE). In a recent study, we introduced a gel-free capillary zone electrophoresis (CZE) method for rapid, high-resolution separation of oligonucleotides at acidic pH.
Results:
Here we optimized a gel-free capillary zone electrophoresis (CZE) method operating at acidic pH using a PVA-coated capillary and malonic acid-ammonia buffer. Adjustments to buffer composition, cation type, and temperature (up to 60 °C) enhanced resolution and reduced current, enabling rapid, high-resolution separation of phosphodiester oligonucleotides ranging from 18 to 25 nucleotides with excellent repeatability (<0.5% mobility variation).
Significance:
This gel-free CZE approach provides a fast and reproducible platform, facilitating detailed characterization of phosphodiester oligonucleotides, microDNA, and microRNA in pharmaceutical and bioanalytical contexts.
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