Development and validation of a method for routine base composition analysis of phosphorothioate oligonucleotides

Area of Science:

• Analytical Chemistry
• Biochemistry
• Oligonucleotide Synthesis

Background:

• Phosphorothioate oligonucleotides are crucial in various biological applications.
• Accurate base composition determination is vital for quality control and research.
• Existing methods may lack precision or accuracy for these modified nucleic acids.

Purpose of the Study:

• To develop and validate a robust method for routine base composition analysis of phosphorothioate oligonucleotides.
• To assess the precision and accuracy of the developed assay.
• To confirm the reliability of the method using orthogonal techniques.

Main Methods:

• Oxidation of phosphorothioate internucleotide linkages using a tetrahydrofuran-water-methylimidazole mixture.
• Enzymatic hydrolysis (enzymolysis) of the oxidized oligonucleotides.
• High-Performance Liquid Chromatography (HPLC) for quantification of individual nucleobases.
• Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) for validation.

Main Results:

• The oxidation step achieved 97.3% effectiveness.
• Inter-day relative standard deviation (RSD) for base composition analysis ranged from 1.1% to 1.3%.
• Inter-lot variation was observed to be between 0.6% and 2.0%.
• MALDI-TOF/MS confirmed the accuracy of the determined nucleobase ratios.

Conclusions:

• A reliable and accurate method for routine base composition determination of phosphorothioate oligonucleotides has been established.
• The assay demonstrates high precision and accuracy, suitable for quality control.
• The developed method, validated by mass spectrometry, provides confidence in oligonucleotide characterization.