Myth busting: “You cannot sequence oligonucleotides over 20 to 30 nucleotides long by LCMS/MS” Learn how to routinely sequence 100 nt oligonucleotides

Importance of the Topic

Oligonucleotide-based therapeutics are rapidly expanding, with applications in gene regulation, vaccines and precision medicine. Reliable sequence verification of long oligonucleotides (up to 100 nucleotides) is critical for identity testing, impurity profiling and regulatory compliance. Traditional LC-MS/MS approaches were limited to short fragments (20–30 nt), creating a gap in quality control for advanced oligonucleotide drugs.

Objectives and Study Overview

This work aimed to develop and demonstrate a robust liquid chromatography–high-resolution mass spectrometry (LC-HRMS) workflow capable of sequencing intact oligonucleotides up to 100 nucleotides. Key goals included optimizing chromatographic conditions to promote high charge states, fine-tuning HCD fragmentation energies for long sequences, and achieving full sequence coverage without enzymatic digestion.

Methodology and Instrumentation

Sample Preparation:

• Synthetic modified RNA and ssDNA oligonucleotides diluted to 1 mg/mL in water.

Chromatography:

• RP-IP separation on a DNAPac RP column (4 µm, 2.1×100 mm) at 50 °C with active pre-heating.
• Eluent A: 20 mM triethylamine (TEA), 80 mM hexafluoroisopropanol (HFIP) in water.
• Eluent B: 20 mM TEA, 80 mM HFIP in acetonitrile or methanol; flow rate 0.3 mL/min.
• High pH (9.5–10.0) to promote higher charge states.

Mass Spectrometry and Data Analysis:

• Thermo Scientific™ Orbitrap Exploris™ 240 in HMR full-MS mode with HCD fragmentation.
• Stepped normalized collision energies (e.g. 13/17/19 or 15/18/21) optimized by oligonucleotide length.
• BioPharma Finder™ 5.1 software for automated fragment annotation and sequence mapping.

Instrumentation Used

• Thermo Scientific™ Vanquish™ Flex Binary UHPLC system
• DNAPac RP column (4 µm, 2.1×100 mm)
• Thermo Scientific™ Orbitrap Exploris™ 240 mass spectrometer
• Thermo Scientific™ BioPharma Finder™ Software 5.1

Main Results and Discussion

Baseline chromatographic separation was achieved for a 6-component ssDNA mix (10–55 nt) and a 100 nt oligonucleotide. High-pH elution produced abundant high charge states, which correlated with improved average structural resolution (ASR). For shorter oligos (10–20 nt), higher HCD energies (up to NCE 21/23/25) provided optimal fragmentation. Conversely, long oligos (50–100 nt) required lower energies (NCE 13/17/19) to avoid over-fragmentation. This approach delivered full sequence coverage (ASR ≈ 1.0) across multiple charge states for a 100 nt target, confirming that direct LC-MS/MS sequencing of large oligonucleotides is feasible.

Benefits and Practical Applications

• Direct sequencing of intact oligonucleotides up to 100 nt without enzymatic digestion.
• Complete sequence validation and impurity detection in a single run.
• Streamlined QC workflow for therapeutic oligonucleotides and mRNA vaccines.
• High throughput and reproducibility using standard UHPLC-MS instrumentation.

Future Trends and Potential Applications

Integration of this LC-HRMS platform with automated sample preparation and data analysis pipelines will further accelerate oligonucleotide drug development. Expansion to heavily modified RNA sequences (e.g. sgRNA, gRNA) and real-time process monitoring are emerging opportunities. Advances in mass analyzer technology and AI-driven spectral interpretation will enhance sensitivity and throughput for next-generation oligonucleotide analytics.

Conclusion

The optimized LC-HRMS method combining high-pH chromatography, high charge states and tailored HCD fragmentation enables routine, direct sequencing of large oligonucleotides (up to 100 nt) with full coverage. This robust workflow addresses a critical analytical challenge in oligonucleotide therapeutics and supports regulatory requirements for sequence validation and impurity analysis.

References

• Vanhinsbergh et al. (2022) Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry, Analytical Chemistry 94(20):7339–7349