Understanding the On-off Retention Mechanism of Oligonucleotides

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A team from the University of Geneva and Bayer AG present their findings on the on-off retention of oligonucleotides and the feasibility of separating them using very short columns.

A recent study published in the Journal of Chromatography A presents an overview of the "on-off" retention mechanism of oligonucleotides in ion-pair reversed-phase liquid chromatography (IP-RPLC) (1). Conducted by researchers from the University of Geneva and Bayer AG, the study aims to present a more thorough understanding of this behavior and the feasibility of rapidly separating oligonucleotides from their impurities using very short columns.

Microscopic illustration of DNA double helix structure, highlighting gene silencing techniques with RNA interference, CRISPR-Cas9, and antisense oligonucleotides, for genetic research applications: © john - stock.adobe.com

Microscopic illustration of DNA double helix structure, highlighting gene silencing techniques with RNA interference, CRISPR-Cas9, and antisense oligonucleotides, for genetic research applications: © john - stock.adobe.com

Oligonucleotides, short DNA or RNA molecules, are at the forefront of modern medicine, and are used to treat a variety of conditions, such as cancers, neurodegenerative diseases, and respiratory disorders. Research in this arena is attracting investment, and it is predicted that 10% of new FDA-approved drugs will be oligonucleotide-based in the future (2). 

IP-RPLC is seen as the gold standard for oligonucleotide analysis. Recent research has focused on the "on-off" retention behavior of oligonucleotides using this technique, where minor changes in the mobile phase's acetonitrile (ACN) concentration can impact the retention. This study aimed to build upon this knowledge by investigating the S values (the slope of the retention model) across a range of mobile phase conditions.

By systematically evaluating key factors—such as ion-pairing (IP) reagent hydrophobicity, IP concentration, mobile phase pH, buffering acids, and column temperature—the team identified optimal conditions for maximizing the S value. In fact, mobile phase conditions were found to influence S values in the following order: IP hydrophobicity > IP concentration > column temperature > buffering acid > mobile phase pH. The findings revealed that conditions minimizing ion-pair interactions, such as using less hydrophobic or lower-concentration IP reagents, enhanced the on-off retention mechanism, making it highly sensitive to ACN levels.

The researchers then developed ultra-fast separation techniques for two therapeutic oligonucleotides: a 20-mer antisense oligonucleotide (ASO) without phosphorothioate (PS) modifications and a large (nearly 100 nucleotide units) single-guide RNA (sgRNA). For the ASO, conditions included a weak ion-pairing reagent (triethylamine, or TEA) and acetate as a buffering acid, with a column temperature of 40 °C. These parameters resulted in a high S value of 87, enabling effective separation of impurities within one to two minutes. For the sgRNA, the team employed a more hydrophobic IP reagent (hexylamine, or HA) and elevated temperatures (80 °C) to achieve the highest possible S values and prevent the separation of diastereomers. This resulted in an S value of 43, which is acceptable for achieving an on-off retention mechanism (an S-value of at least 30 is required when using ultra-short columns). Both ASO and sgRNA were separated in under one minute using a 5- mm column.

The presented method offers an acceleration and aid to the development of oligonucleotide-based drugs, which aligns with the growing reliance of them in modern medicine. The ability to rapidly assess purity and quality will be critical for scaling up production and ensuring the safety and efficacy of new treatments.

References

(1) Lardeux, H.; Bagci, S.; Gao, M.; et al. Understanding the Fundamentals of the On-off Retention Mechanism of Oligonucleotides and their Application to High Throughput Analysis. J. Chromatogr. A 2025, 1739, 465523. DOI: 10.1016/j.chroma.2024.465523

(2) Roberts, T. C.; Langer, R.; Wood, M. J. A. Advances in Oligonucleotide Drug Delivery. Nat. Rev. Drug Discov.2020, 19, 673–694. DOI: 10.1038/s41573-020-0075-7

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