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Separation of modified 2'-deoxyoligonucleotides using ion-pairing reversed-phase HPLC
Stacy L Gelhaus1, William R LaCourse
1Department of Chemistry and Biochemistry, University of Maryland, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Summary
Ion-pairing reversed-phase liquid chromatography (IP-RPLC) effectively separates oligonucleotides with distinct alkyl modifications. This method reveals how base identity, adduct position, and length influence retention, aiding modified oligonucleotide purification.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Chromatography
Background:
- Modified oligonucleotides are crucial in diagnostics and therapeutics.
- Understanding the impact of chemical modifications on oligonucleotide properties is essential for their development and application.
- Ion-pairing reversed-phase liquid chromatography (IP-RPLC) is a powerful technique for separating complex mixtures.
Purpose of the Study:
- To investigate the effects of varying alkyl modifications on oligonucleotide retention using IP-RPLC.
- To determine how different alkyl adducts influence the chromatographic behavior of oligonucleotides.
- To demonstrate the utility of IP-RPLC for purifying modified oligonucleotides.
Main Methods:
- Synthesis of 18-mer oligonucleotides with identical base sequences but differing alkyl modifications.
- Analysis of these modified oligonucleotides using ion-pairing reversed-phase liquid chromatography (IP-RPLC).
- Evaluation of retention times and chromatographic selectivity based on alkylation patterns.
Main Results:
- IP-RPLC successfully distinguished between oligonucleotides differing by single alkyl modifications.
- Oligonucleotide retention was found to be dependent on the identity of the nucleobase, and the position and length of the alkyl adduct.
- Observed separation phenomena were attributed to changes in charge and hydrophobicity induced by alkylation.
Conclusions:
- IP-RPLC provides high resolution for separating oligonucleotides with subtle structural variations.
- The technique is valuable for characterizing and purifying chemically modified oligonucleotides.
- Chromatographic selectivity in IP-RPLC significantly enhances the purification of modified oligonucleotides.
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