MAP SEQUENCE - A NOVEL SOFTWARE APP FOR RAPID AND AUTOMATED SEQUENCE MAPPING OF mRNA MOLECULES

Significance of the topic

The mapping and sequence confirmation of therapeutic mRNAs is critical for quality control, identity testing, and characterization of sequence integrity and modifications. Rapid, high-confidence workflows that minimize manual interpretation—combining optimized enzymatic digestions, high-resolution mass spectrometry, and tailored informatics—are essential for accelerating development and ensuring manufacturing consistency of nucleic-acid-based therapeutics.

Objectives and overview of the study

This work presents MAP Sequence, a software application (App) integrated with the waters_connect SYNTHETIC Library, designed to automate and accelerate sequence mapping of mRNA molecules. The study demonstrates the combined use of novel RNase reagents (RapiZyme MC1 and Cusativin), LC-MS data-independent acquisition (MSE) on the high-resolution Xevo MRT QTof mass spectrometer, and dedicated informatics to achieve near-complete sequence coverage and automated resolution of isomeric digestion products for a model 1.9 kb firefly luciferase (Fluc) mRNA.

Methods and workflow

• Sample: In vitro transcribed Fluc mRNA with Cap1, ~1,919 nt plus poly(A) tail.
• Enzymatic digestion: Parallel digests with RNase T1 and two recombinant RNase T2-family enzymes, RapiZyme MC1 (5'-uridine preference) and RapiZyme Cusativin (3'-cytidine preference). Enzyme concentrations and digestion times were tuned to generate informative missed cleavages and longer unique fragments.
• Chromatography: Ion-pair reversed-phase UHPLC (ACQUITY Premier OST column, 2.1 × 150 mm) with dipropylamine (DPA/DPD) and HFIP buffers; gradient 0→50% B over 60 min at 0.4 mL/min, column at 60 °C; UV at 260 nm.
• Mass spectrometry: Data-independent acquisition (MSE) on Xevo MRT (multi-reflectron TOF) QTof; 0.5 s scans over 400–4000 Da; low-energy CE 6 V, high-energy CE ramp 30–55 V.
• Informatics: waters_connect platform with SYNTHETIC Library App v2.0 to generate in-silico digests and MAP Sequence App v2.0 for automated assignment of precursor and fragment data, isomer discrimination, and report generation. Typical processing used a 5 ppm search tolerance; instrument mass accuracy routinely <1 ppm.

Instrumentation used

• Xevo MRT QTof Mass Spectrometer (Waters)—high-resolution multi-reflectron TOF analyzer.
• ACQUITY Premier UPLC BSM System with ACQUITY Premier OST column (2.1 × 150 mm).
• waters_connect Informatics Platform with SYNTHETIC Library App and MAP Sequence App.

Main results and discussion

• Sequence coverage: Combining digests from RapiZyme MC1 and Cusativin with RNase T1 produced near-complete Fluc mRNA sequence coverage of 95.4% (assuming up to four missed cleavages), demonstrating that multi-enzyme strategies markedly improve coverage versus single-enzyme workflows.
• Mass spectrometry performance: Xevo MRT delivered high resolving power (>100,000 FWHM), sub-ppm mass accuracy for both precursors and fragments, and sufficient sensitivity to assign the majority of chromatographic signal to matched digestion products. Typical precursor isotopic envelopes and charge-state series (e.g., -2 to -10) were resolved and used for confident assignments.
• Automated assignment and isomer resolution: MAP Sequence automated matching of low-energy precursors and elevated-energy fragment ions (MSE) to assign digestion products. The App can automatically accept or reject isomeric candidates based on fragment-ion coverage criteria (example: one 12-mer showed 100% fragment coverage and was confirmed; its isomer had only 50% coverage and was rejected).
• Enzyme specificity and strategy: RapiZyme MC1 (uridine-biased) and Cusativin (cytidine-biased) produce longer, more unique digestion products than RNase T1 in many contexts, especially when controlled missed cleavages are induced by tuning enzyme amount and digestion time. This feature reduces ambiguity in assignments and lessens reliance on extensive manual MS2 interpretation.

Benefits and practical applications

• Higher confidence sequence confirmation for long mRNA constructs through complementary enzyme panels and high-resolution DIA data.
• Reduced manual curation: Automated assignment and isomer discrimination save analyst time and improve reproducibility.
• Regulatory and manufacturing suitability: The workflow is implemented on a compliance-ready informatics platform, supporting potential use in QC, batch release, and process development for mRNA therapeutics.
• Scalability: The combination of standardized LC-MS acquisition and software-assisted processing supports routine adoption in analytical laboratories focused on nucleic-acid therapeutics.

Future trends and potential applications

• Extension to modified nucleotides: Incorporation of workflows and library entries to detect and localize common base or backbone modifications in therapeutic mRNAs.
• Throughput and automation: Shorter gradients, automated digestion robotics, and batch processing in waters_connect could enable higher sample throughput and integration into release testing pipelines.
• Expanded enzyme toolboxes: Additional sequence-specific RNases and controlled digestion chemistries will further improve unique fragment generation and coverage for diverse sequences.
• AI-assisted interpretation: Machine learning could refine fragment-to-sequence assignment, detect subtle modification signatures, and predict optimal enzyme panels for new constructs.
• Standardization: Harmonized protocols and acceptance criteria across labs to facilitate cross-site comparability and regulatory acceptance of LC-MS-based mRNA sequence mapping.

Conclusion

Combining novel RNase reagents (RapiZyme MC1 and Cusativin), high-resolution DIA (MSE) on the Xevo MRT QTof, and the MAP Sequence/SYNTHETIC Library informatics provides an efficient, automated route to high-confidence sequence mapping of long mRNA molecules. The workflow achieves near-complete coverage, robust isomer discrimination, and reduced manual interpretation, making it attractive for research and quality functions in mRNA therapeutic development and manufacturing.

References

1. Waters application note: Tunable Digestion of RNA Using RapiZyme RNases to Confirm Sequence and Map Modifications, 2024, P/N 720008539EN.
2. Waters application note: RNA Digestion Product Mapping Using an Integrated UPLC-MS and Informatics Workflow, 2024, P/N 720008553EN.
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. doi:10.1016/j.pep.2021.105987.
4. Thakur P, Atway J, Limbach PA, Addepalli B. RNA Cleavage Properties of Nucleobase-Specific RNase MC1 and Cusativin Are Determined by the Dinucleotide-Binding Interactions in the Enzyme-Active Site. Int J Mol Sci. 2022;23:7021.
5. Waters application note: Sequence Mapping of mRNA Digests Using the Xevo MRT Mass Spectrometer and waters_connect MAP Sequence 2.0 Application, 2025, P/N 720009171EN.