Oligonucleotide Characterization and Separation Techniques

Characterisation and separation of oligonucleotides underpin their development as therapeutics and research tools. Oligonucleotides are short nucleic acid sequences whose structural complexity—stemming from variations in length, backbone modifications and stereochemistry—presents analytical challenges. Ion-pair reversed-phase liquid chromatography remains the workhorse for profiling length variants and backbone impurities, with advances in stationary phases, ion-pair reagents and gradient design improving resolution of closely related species such as phosphorothioate diastereomers. Hydrophilic interaction chromatography has emerged as a complementary approach for highly polar oligonucleotides, offering alternative selectivity when coupled to mass spectrometry. Ion-exchange formats provide a robust route to assess longer molecules and support preparative purification. Innovations in column hardware, from superficially porous particles to bioinert materials, have minimised non-specific adsorption and enhanced on-column stability. Together, these techniques enable precise determination of purity, stereochemical configuration and sequence integrity, facilitating global quality control and regulatory compliance in oligonucleotide drug development and molecular biology applications.

Research from Nature Portfolio

A foundational report demonstrated that conventional ion-pair reversed-phase chromatography is suboptimal for long synthetic RNAs such as single-guide RNAs used in genome editing. By comparing high-temperature IP-RP with alkaline ion-exchange, the study revealed superior resolving power and on-column stability under denaturing IEX conditions. This approach allowed accurate purity evaluation of 100-nucleotide RNAs and established protocols to assess sample integrity during chromatography. The work set a benchmark for quality control of various RNA modalities, including sgRNA, tRNA and mRNA, by exploiting the robustness of ion-exchange mobile phases and optimising column conditions for long sequences.

Oligonucleotide Characterization and Separation Techniques publication trend

The graph below shows the total number of articles in oligonucleotide characterization and separation techniques across all publications each year (not limited to Nature Index journals).

Technical terms

Oligonucleotide: A short polymer of nucleotides used in therapeutics and molecular biology.

Ion-pair reversed-phase liquid chromatography (IP-RPLC): A separation technique in which ionic analytes are paired with a counter-ion in the mobile phase to enhance interaction with a hydrophobic stationary phase.

Hydrophilic interaction chromatography (HILIC): A chromatographic mode that retains polar compounds by partitioning between a water-enriched layer on the stationary phase and the mobile phase.

Ion-exchange chromatography (IEX): A method that separates analytes based on their net charge by reversible binding to oppositely charged groups on the stationary phase.

Diastereomer: One of a pair of stereoisomers that are not mirror images, often arising in phosphorothioate oligonucleotides.

Ion-pair reagent: A charged additive in the mobile phase that forms ion pairs with analytes to modify retention and selectivity in chromatography.

References

1. Recent advances and current challenges in hydrophilic interaction chromatography for the analysis of therapeutic oligonucleotides. TrAC Trends in Analytical Chemistry (2024).
2. Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides. Analytical and Bioanalytical Chemistry (2019).
3. HPLC methods for purity evaluation of man-made single-stranded RNAs. Scientific Reports (2019).
4. Nucleic acid separations using superficially porous silica particles. Journal of Chromatography A (2016).
5. Separation of therapeutic oligonucleotides using ion-pair reversed-phase chromatography based on fundamental separation science. Journal of Chromatography Open (2023).
6. Bioanalysis of Oligonucleotide by LC–MS: Effects of Ion Pairing Regents and Recent Advances in Ion-Pairing-Free Analytical Strategies. International Journal of Molecular Sciences (2022).
7. The impact of low adsorption surfaces for the analysis of DNA and RNA oligonucleotides. Journal of Chromatography A (2022).
8. Impact of stationary-phase pore size on chromatographic performance using oligonucleotide separation as a model. Journal of Chromatography A (2020).