Characterization of a synthetic double stranded siRNA using high-resolution mass spectrometry

Importance of Topic

Therapeutic oligonucleotide therapeutics such as antisense oligonucleotides and small interfering RNA have shown growing success in treating a variety of diseases. Accurate separation, identification, and mapping of these molecules and their impurities are vital in research, development, and quality control.

Objectives and Overview of the Study

This study demonstrates a high-resolution mass spectrometry method coupled to ion-pair reversed-phase liquid chromatography to separate and characterize a synthetic double-stranded siRNA and its diastereomers in a single workflow. A combination of UHPLC, Orbitrap mass spectrometry, and advanced data-dependent tandem MS was used to achieve confident identification and mapping of both sense and antisense strands.

Instrumentation Used

• Orbitrap Exploris 120 mass spectrometer
• Vanquish Horizon UHPLC system
• DNAPac RP column (4 μm, 2.1 × 50 mm)
• BioPharma Finder software version 4.1

Methodology

Ion-pairing reversed-phase LC was performed using two solvent systems: HFIP with DIPEA at pH ~8 and HFIP with TEA at pH ~9. Gradients and column temperatures (30–70 °C) were adjusted to evaluate duplex denaturing and non-denaturing conditions. MS acquisition employed high-resolution accurate mass full scans followed by data-dependent MS2 (Top4) using stepped collision energies. Sequence mapping was conducted in BioPharma Finder using the oligonucleotide analysis workflow.

Main Results and Discussion

Using DIPEA solvents at 70 °C, the siRNA duplex denatures into single sense and antisense strands, each detected as separate chromatographic peaks with high-quality MS2 spectra and full sequence coverage. Under TEA solvents at pH ~9 and lower temperatures (30–50 °C), the non-denaturing duplex produces two distinct peaks corresponding to diastereomeric configurations arising from a phosphorothioate chiral center. The identities of all species were confirmed by high-resolution mass accuracy (<2 ppm) and complete fragment mapping.

Benefits and Practical Applications

• Simultaneous separation and mapping of sense, antisense, and diastereomeric duplex species in a single analysis.
• High resolution accurate mass and data-dependent MS2 enable confident identification and impurity characterization.
• Applicable to therapeutic siRNA development, quality control, and impurity profiling.

Future Trends and Possibilities

Advancements may include integration of ion mobility, real-time AI-driven data interpretation, and expansion to more heavily modified oligonucleotides. These developments will further enhance throughput and depth of structural characterization in oligonucleotide research.

Conclusion

The combined UHPLC-HRMS platform and data-dependent tandem MS strategy provide a robust workflow for comprehensive characterization of double-stranded siRNA and its diastereomers. This approach offers high confidence in sequence mapping and impurity detection, supporting therapeutic oligonucleotide development and quality assurance.

Reference

1. Bajan S et al. RNA-based therapeutics: from antisense oligonucleotide to miRNAs. Cells 2020;9:137.
2. Sutton JM et al. Current state of oligonucleotide characterization using LC-MS. J Am Soc Mass Spectrom 2020;31:1775.
3. Pourshahian S. Therapeutic oligonucleotides, impurities, and degradation products. Mass Spectrom Rev 2019;doi:10.1002/mas.21615.
4. Liu HC et al. Oligonucleotide mapping using BioPharma Finder software. Thermo Scientific Application Note 73789;2020.
5. Liu HC et al. Identification and quantitation of oligonucleotides and impurities. Thermo Scientific Application Note 73870;2020.