mRNA direct sequence mapping using automated partial digestion with magnetic nuclease and LC-HRMS

Importance of the Topic

mRNA therapeutics and vaccines have rapidly emerged, exemplified by COVID-19 vaccines.
Accurate sequence mapping of large mRNA molecules is critical to ensure identity, integrity, and detect impurities.
Direct LC-HRMS analysis enables rapid, reliable characterization without conversion to cDNA.

Objectives and Study Overview

This work aims to develop an automated, reproducible LC-MS workflow for direct sequencing of large mRNAs using controlled partial RNase T1 digests on magnetic beads.
It seeks to generate large sequence-specific fragments, optimize chromatographic separation, and automate data annotation to achieve comprehensive sequence coverage (>85%) for modified and unmodified mRNAs.

Methodology and Instrumentation

• Sample Preparation: Partial digestion of 20-40 µg mRNA with immobilized RNase T1 on magnetic beads at 37-60 °C for 5-30 min; beads removed to stop reaction.
• Chromatography: Ion-pair reverse-phase UHPLC on a DNAPac RP column with TEA/HFIP buffer, 60 °C, gradient over 60 min.
• Mass Spectrometry: Thermo Scientific Orbitrap Exploris 240; negative ion DDA MS1 (450-3000 m/z), MS2 (150-2000 m/z), resolutions 120k/30k; stepped HCD collision energy.
• Data Analysis: BioPharma Finder 5.0 for oligonucleotide mapping; mass accuracy ≤10 ppm; confidence ≥90%; average structural resolution ≤2.0; MS/MS fragmentation matching.

Results and Discussion

• Partial RNase T1 digests reproducibly generated 10-50 nt fragments; reproducibility: eGFP mRNA coverage 70.6% ±1.7% across replicates.
• High-resolution chromatography separated isomeric oligonucleotides, enabling unique sequence assignments up to 50 nt.
• Direct mapping of SARS CoV-2 spike mRNA and eGFP mRNA yielded >86% coverage; excluding polyA tail gave up to 96%.
• Modified mRNA containing 5-methoxyuridine was sequenced, achieving >90% coverage; MS/MS spectra distinguished modified from unmodified fragments.

Benefits and Practical Applications

• Rapid 90-minute workflow suitable for QC environments.
• High confidence in sequence identity and integrity for large, modified mRNAs.
• Automation reduces variability and prevents post-injection digestion on LC column.
• Applicable to vaccine development, therapeutic mRNA validation, and impurity profiling.

Future Trends and Opportunities

• Integration with QC release testing in biopharmaceutical manufacturing.
• Expansion to other nucleic acid therapeutics, including siRNA and long noncoding RNAs.
• Enhancements in software algorithms for de novo sequencing of unknown RNA modifications.
• Automation of sample prep for high-throughput screening.

Conclusion

A semi-automated LC-HRMS workflow combining partial RNase T1 digests with high-resolution MS and automated data analysis enables direct, high-coverage sequencing of large mRNA molecules.
This method supports fast, reliable identity testing of modified and unmodified mRNA therapeutics.

Reference

No literature references were provided in the original document.