A versatile reversed phase platform for short, intermediate, and long nucleic acid analysis

Importance of the topic

In modern biopharmaceutical research and quality control, the ability to efficiently analyze nucleic acids across a broad size range is critical. Therapeutic classes such as siRNA, gRNA used in CRISPR/Cas9, and long mRNA vaccines demand reliable separation methods to ensure purity and performance.

Objectives and overview of the study

This study evaluates the performance of the DNAPac RP reversed-phase column for separating nucleic acids from 10 to 10,000 nucleotides. Comparisons are made against conventional C18 silica columns to highlight improvements in resolution, speed, and robustness.

Methodology and instrumentation

The experiments utilized a Thermo Scientific Vanquish Horizon UHPLC system equipped with a DNAPac RP column (4 µm, 50–2000 Å polymeric supermacroporous resin). An ion-pairing mobile phase consisting of 0.1 M triethylammonium acetate (A) and acetonitrile (B) was employed under gradient elution. Key parameters included temperatures up to 60 °C, flow rates of 0.4 mL/min, and UV detection at 260 nm. The robustness of the polymeric resin was demonstrated by its tolerance to pH 0–14 and temperatures up to 100 °C.

Key results and discussion

The DNAPac RP column achieved baseline separation of:

• Short oligonucleotides (12–40 mer) within a 9 min gradient, all eight components resolved.
• Intermediate lengths (98–100 mer) with sharper, more defined peaks compared to 2.5 µm and 1.7 µm C18 columns.
• Long fragments (67–1353 bp) from ΦX174-BsuRI digests in 12 min, unattainable by conventional columns due to pore limitations.

The broad pore distribution (50–2000 Å) confirmed by BET and SEM analyses underpins the column’s superior performance for a wide molecular weight range.

Benefits and practical applications

The DNAPac RP platform offers:

• Versatile separation of nucleic acids from small siRNA to long mRNA and DNA fragments.
• Enhanced resolution and peak sharpness, reducing analysis time.
• High stability under extreme pH and temperature conditions, facilitating method flexibility.

This makes it well suited for therapeutic oligonucleotide QC, impurity profiling, and high-throughput screening workflows.

Future trends and potential applications

Emerging opportunities include:

• Integration with mass spectrometry for sequence mapping of large RNA therapeutics.
• Automation in high-throughput platforms for next-generation nucleic acid analyses.
• Development of tailored stationary phases for complex nucleic acid conformations and modifications.

Conclusion

The DNAPac RP column provides a robust, high-resolution reversed-phase solution for nucleic acid analysis across a broad size spectrum, outperforming traditional C18 silica columns. Its stability and versatility position it as a valuable tool in research and industrial laboratories.

References

1. Vanhinsbergh C.J.; Criscuolo A.; Sutton J.N.; Murphy K.; Williamson A.J.K.; Cook K.; Dickman M.J. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. Analytical Chemistry 2022, 94, 7339–7349.
2. Wei B.; Wang J.; Cadang L.; Goyon A.; Chen B.; Yang F.; Zhang K. Development of an Ion-Pairing Reversed-Phase Liquid Chromatography–Mass Spectrometry Method for Characterization of CRISPR Guide Ribonucleic Acid. Journal of Chromatography A 2022, 1665, 462839.
3. Packer M.; Gyawali D.; Yerabolu R.; Schariter J.; White P. A Novel Mechanism for the Loss of mRNA Activity in Lipid Nanoparticle Delivery Systems. Nature Communications 2021, 12, 6777.