New Research Explores Role of Nucleotide Hydrophobicity in Oligonucleotide Separation

Researchers from Waters Corporation in Milford, Massachusetts and BioSpring in Frankfurt, Germany investigated how nucleotide hydrophobicity impacts oligonucleotide (ON) separation in liquid chromatography. Their findings were published in the Journal of Chromatography A (1).

ON therapeutics have revolutionized precision medicine, offering highly specific and versatile modalities for treating various diseases. Antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and aptamers are able to modulate gene expression by degrading target genes with specific mechanisms or bind molecular targets specifically and modulate downstream biochemical pathways. Other ON-based therapies, like messenger RNA (mRNA)-based vaccines or clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques, have shown potential for future applications. Therapeutic ONs have the potential to change standards of care for many diseases, with current advancements enabling improved product stability and optimizing delivery systems to reach therapeutic targets (2).

ON synthesis is prone to generating impurities arising from incomplete coupling reactions, premature truncation, or side reactions during elongation. The methods of choice for analyzing synthesis-related impurities and diastereomers are high-performance liquid chromatography (HPLC) and ultra high-performance liquid chromatography (UHPLC) (1). These techniques greatly assist with ON purification and characterization, including ion-pairing reverse-phase (IP RP) liquid chromatography (LC). The separation power of IP RP LC is profoundly influenced by the choice of ion-pairing reagents, solvents, and buffering acids. Optimizing separation parameters is vital for high resolving power of the selected LC methods.

In this study, the scientists investigated the contribution of nucleotide (nt) type and modifications on the retention and resolution of 22–24 nt long oligonucleotides in ion-pairing reversed-phase (IP RP) liquid chromatography (LC) and hydrophilic interaction chromatography (HILIC) methods. The nucleotides’ relative hydrophobicity can affect the oligonucleotide LC retention and separation selectivity. The ion-pairing reagents of higher hydrophobicity require greater organic solvents concentration to elute oligonucleotides in IP RP LC.

The scientists observed that higher concentrations of organic solvent reduced the contribution of (oligo)nucleotide hydrophobicity to retention, while also leading to more uniform separation of full-length ONs from their truncated products. Further, they also investigated the diastereomeric resolution of 24 nt oligonucleotides with a single phosphorothioate modification at 5′-end. The separation is nucleotide-motif dependent; G (and modified G) nucleotides adjacent to the phosphorothioate linkage yield higher resolution of isomers than other types of nucleotides in IP RP LC and HILIC.

The scientists hoped to understand the separation of modified ONs from their n-x impurities. They noticed several factors; for example, retaining 23 nt ONs of the same core sequence and different terminal (modified) nucleotides confirms that chemical modifications of nucleotides contribute to IP RP LC. IP RP LC and HILIC show inversed retention correlation for tested ONs when using relatively polar DPA ion pairing systems.

These findings and experiments continue the trend of analyzing ONs in different fashions. This study is just one of many set to broaden humanity’s understanding of ONs, so that they can be harnessed to their full potential.

References

(1) Gilar, M.; Schomann, N.; Schott, S.; Rühl, M. Impact of Nucleotide Hydrophobicity on Oligonucleotides Separation in Liquid Chromatography. J. Chromatogr. A 2025, 1753, 465968. DOI: 10.1016/j.chroma.2025.465968

(2) Croguennec, A. Oligonucleotide Therapeutics: A Beginner’s Guide. Thermo Fisher Scientific 2023. https://www.thermofisher.com/blog/analyteguru/oligonucleotide-therapeutics-a-beginners-guide/ (accessed 2025-6-17)

(3) Matheson, A. HPLC 2025 Day 1: Oligomer Separation Sessions. LCGC International 2025. https://www.chromatographyonline.com/view/hplc-2025-update-oligomer-separations (accessed 2025-6-17)