RNA Sequence Mapping Via Endonuclease Digestion and Lc-MS Analysis Via Novel Informatics Workflows

Importance of the topic

Advances in RNA therapeutics demand rigorous analytical workflows to confirm sequence integrity, modifications and full coverage. Accurate mapping of synthetic oligonucleotides, guide RNAs (sgRNA) and mRNA constructs underpins quality control in biopharmaceutical development and regulatory compliance.

Objectives and Study Overview

This study presents an integrated liquid chromatography–mass spectrometry (LC-MS) and informatics workflow for endonuclease digestion mapping of a 100-mer sgRNA standard and a 1 019-nucleotide mRNA encoding GFP. The goals were to achieve complete sequence coverage, resolve structural isomers and streamline data processing using novel Waters software applications.

Methodology and Instrumentation

The analytical platform combined an ACQUITY Premier UHPLC with a Xevo MRT QTof mass spectrometer operated in data-independent acquisition (MSE) mode. Key parameters included:

• Column: ACQUITY Premier OST, 1.7 μm, 2.1×150 mm
• Mobile phases: 10 mM n-dipropylamine/40 mM HFIP in water (pH 8.6) and in 50 % methanol
• Gradient: 0–50 % organic over 45 min at 0.3 mL/min, 60 °C
• MS settings: 340–4 000 Da mass range; low-energy CE 6 V; high-energy CE ramp 30–55 V; ~80 000 resolving power

Endonucleases: RNase T1 (100 U/µL) and RapiZyme MC1 (200 mM ammonium acetate, pH 8.0). Digests were analyzed immediately with 5 µL injections.

Informatics applications on the waters_connect Platform 4.1 included:

• SYNTHETIC Library App for in silico digestion and coverage prediction
• MAP Sequence App for precursor assignment from MS1 data
• CONFIRM Sequence App for structural isomer elucidation using MSE fragmentation

Main Results and Discussion

Complete sequence coverage (100 %) of the 100-mer sgRNA was achieved using RapiZyme MC1, surpassing RNase T1 alone. The mRNA digest yielded >85 % coverage per enzyme, and combining RNase T1 and MC1 predictions approached 99.9 %. The high-resolution Xevo MRT QTof enabled mass accuracy within ±3 ppm, facilitating unambiguous oligonucleotide identification. CONFIRM Sequence App successfully resolved isomeric digestion products by comparing dot-map representations and MS/MS fragments.

Benefits and Practical Applications

These workflows provide:

• High confidence in sequence mapping for sgRNA and mRNA therapeutics
• Improved mass resolution and sensitivity for low-abundance fragments
• Automated data processing to reduce manual interpretation
• Capability to track modifications and confirm phosphorothioate positions

Future Trends and Possibilities

Further developments may include expanded enzyme panels for even higher coverage, integration of MS3 fragmentation for deeper structural insights, and machine-learning algorithms to predict optimal digestion conditions. Continuous informatics enhancements will support real-time release testing and in-line quality control in RNA manufacturing.

Conclusion

The combined LC-MS and waters_connect informatics workflow delivers comprehensive RNA sequence mapping with high accuracy and automation. This approach is well suited for research, development and quality assurance of RNA-based therapeutics.

References

1. Waters Corporation. Tunable Digestion of RNA Using RapiZyme RNases to Confirm Sequence and Map Modifications; Application Note P/N 720008539EN, 2024.
2. Waters Corporation. RNA Digestion Product Mapping Using an Integrated UPLC-MS and Informatics Workflow; Application Note P/N 720008553EN, 2024.
3. Grunberg S, Wolf EJ, Jin J, Ganatra MD, Becker K, Ruse C, Taron CH, Correa IR, Yigit E. Enhanced Expression and Purification of Nucleotide-specific Ribonucleases MC1 and Cusativin. Protein Expr Purif, 2022;190:105987.
4. Thakur P, Atway J, Limbach PA, Addepalli B. RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin. Int J Mol Sci, 2022;23:7021.
5. Waters Corporation. CONFIRM Sequence: A waters_connect Application for Sequencing of Synthetic Oligonucleotides and Their Impurities; Application Note P/N 720007677EN, 2022.