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Characterization and sequence verification of thiolated deoxyoligonucleotides used for microarray construction.
Arthur Van Aerschot1, Jef Rozenski2
1Rega Institute for Medical Research, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.
Journal of the American Society for Mass Spectrometry
|August 11, 2006
Summary
High purity thiolated deoxyoligonucleotides are essential for microchip fabrication. This study identified and characterized synthesis impurities, revealing modified thiol groups and oxidation products, aiding protocol optimization.
Area of Science:
- Chemical Synthesis
- Biochemistry
- Materials Science
Background:
- Thiolated deoxyoligonucleotides are crucial for microchip fabrication.
- High purity is required to prevent synthesis side products from interfering with applications.
- Previous analyses did not fully characterize these impurities.
Purpose of the Study:
- To identify and characterize impurities formed during thiolated deoxyoligonucleotide synthesis.
- To understand the nature of the modification of the thiol group in side products.
- To provide insights for optimizing synthesis protocols.
Main Methods:
- High-performance liquid chromatography (HPLC) for separation.
- Electrospray mass spectrometry (MS/MS) for identification and characterization.
- Acrylamide derivatization to assess free thiol group presence.
Main Results:
- Impurities were identified that were not standard synthesis failure sequences.
- Side products showed no reaction with acrylamide, indicating a modified thiol group.
- A major impurity was a 5' sulfinic acid containing molecule (45% of a 25-mer).
- Dithio-linked oxidation products were present at 1-5% in 10-mer and 15-mer oligonucleotides.
Conclusions:
- The study successfully identified and characterized key impurities in thiolated deoxyoligonucleotide synthesis.
- Understanding these impurities, such as sulfinic acid and oxidation products, is vital for quality control.
- The analytical methods employed were effective in guiding the optimization of synthesis protocols for higher purity oligonucleotides.