Oligo Mapping of mRNA Digests Using a Novel Informatics Workflow

Significance of the Topic

Advances in mRNA therapeutics require robust analytical methods to confirm sequence integrity and critical quality attributes such as 5′ capping and base modifications. Traditional sequencing approaches lack direct detection of chemical modifications and often require orthogonal confirmation. LC-MS based oligonucleotide mapping paired with automated informatics offers high specificity, sensitivity, and the ability to detect backbone and base alterations, enabling comprehensive characterization of complex mRNA products.

Objectives and Study Overview

This application note presents an end-to-end workflow for mRNA sequence mapping. It integrates novel RNase T2 enzymes (RapiZyme MC1 and Cusativin) with UPLC-QTof MS and automated data processing using the waters_connect MAP Sequence App. The study evaluates sequence coverage, assignment confidence, and 5′ capping efficiency for a 1,019-nucleotide GFP mRNA construct.

Methodology and Instrumentation

Sample Preparation and Enzymatic Digestion
• GFP mRNA was synthesized with a Cap1 structure, denatured, and digested separately with RNase T1, RapiZyme MC1, and RapiZyme Cusativin under controlled buffer and temperature conditions.

LC-MS Acquisition
• System: ACQUITY Premier UPLC coupled to Xevo G3 QTof MS in ESI(–) MSE mode.
• Column: Oligonucleotide BEH C18, 1.7 μm, 2.1×150 mm at 60 °C, flow rate 300 μL/min.
• Mobile phases: 10 mM dipropylethylamine and 40 mM HFIP in water (A) or 50% MeOH (B), pH 8.5.
• MS settings: range m/z 340–4000, capillary voltage 2.5 kV, desolvation 550 °C.

Data Processing
• Predicted digests generated by mRNA Cleaver MicroApp.
• Automated MS1 matching via MAP Sequence App.
• Coverage visualization in Coverage Viewer MicroApp.
• Ambiguities resolved using CONFIRM Sequence App with elevated-energy fragmentation.

Main Results and Discussion

Sequence Coverage Comparison
• RNase T1 achieved ~60% unique coverage due to frequent G-specific cleavage and resultant isomeric products.
• RapiZyme MC1 and Cusativin produced longer and more unique oligonucleotides, yielding ~97% and ~85% coverage respectively.

Assignment Ambiguity Resolution
• Structural isomers (e.g., two 16-mer sequences) with identical masses were distinguished by matching MSE fragment ions in CONFIRM Sequence.

5′ Capping Efficiency Analysis
• MC1 and Cusativin generated a 13-mer digest retaining the 5′ structure, enabling quantification of uncapped and capped species (capping efficiency ~79.8%).
• RNase T1 produced too short uncapped products for retention, precluding efficiency measurement.

Benefits and Practical Applications

• Streamlined automated workflow reduces manual curation and accelerates mRNA sequence confirmation.
• Novel RNase T2 enzymes enhance sequence coverage by generating unique, overlapping oligonucleotides.
• Combined LC-MS and informatics enable simultaneous analysis of sequence integrity and 5′ capping efficiency.

Future Trends and Opportunities

• Integration of MSE fragmentation matching into automated apps for fully untargeted assignment.
• Expansion of the RNase toolkit to cover diverse cleavage specificities and modified nucleotides.
• High-throughput, flow-through digestion approaches for rapid quality control of therapeutic mRNAs.
• Enhanced interoperability with large-language models for real-time data interpretation and reporting.

Conclusion

The combined use of novel RNase T2 enzymes, UPLC-QTof MS, and waters_connect informatics delivers high-confidence mRNA oligo mapping and 5′ cap analysis. This automated workflow significantly improves sequence coverage, reduces ambiguity, and supports comprehensive characterization of mRNA therapeutics.

References

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