Single-based resolution for oligodeoxynucleotides and their phosphorothioate modifications by replaceable capillary...

Area of Science:

• Analytical Chemistry
• Biochemistry
• Molecular Biology

Background:

• Separating short DNA sequences, particularly those with chemical modifications like phosphorothioate (PS-ODNs), presents analytical challenges.
• Capillary gel electrophoresis (CGE) is a powerful technique for DNA analysis, but optimizing conditions for modified oligonucleotides is crucial.

Purpose of the Study:

• To develop and optimize a replaceable capillary gel electrophoretic (CGE) method for resolving mixtures of single-stranded oligodeoxynucleotides (ODNs).
• To differentiate between standard phosphodiester ODNs (PO-ODNs) and their phosphorothioate (PS-ODN) counterparts that differ by a single base.

Main Methods:

• Utilized a replaceable capillary gel electrophoresis system with polyethylene glycol (PEG) 35000 as the sieving matrix.
• Investigated the impact of acetonitrile as a separation additive and varied temperature and urea concentrations.

Main Results:

• The developed method successfully separated model compounds of 18-20 mer PO-ODNs and PS-ODNs with identical sequences differing by a single base.
• Polyethylene glycol (PEG) 35000 demonstrated a lower influence on PS-ODN resolution compared to PO-ODNs.
• Acetonitrile significantly enhanced separation resolution, while temperature had a more pronounced effect than urea.

Conclusions:

• A robust and optimized replaceable CGE method was established for distinguishing between PO-ODNs and PS-ODNs.
• The findings highlight the utility of PEG 35000 and acetonitrile in improving the separation of modified oligonucleotides.
• Temperature control is a critical factor for achieving high-resolution separation in this CGE system.