Tunable Digestions of RNA Using RapiZyme™ RNases to Confirm Sequence and Map Modifications

Importance of the Topic

The development of RNA therapies such as sgRNA and mRNA vaccines has accelerated the need for reliable analytical methods. Oligonucleotide mapping by LC-MS provides direct detection of RNA fragments and modified nucleosides, ensuring identity, purity and sequence integrity.

Objectives and Study Overview

This application note evaluates two recombinant RNases, RapiZyme MC1 and Cusativin, for their ability to generate complementary RNA fragments for robust sequence coverage of sgRNA. The study compares these enzymes with traditional RNases T1 and 4 and integrates informatics workflows for streamlined data analysis.

Methodology and Instrumentation

• Sample Preparation: sgRNA denaturation and digestion in RNase-free conditions using optimized buffers without chemical denaturants.
• Enzymes: RapiZyme MC1 (pU cleavage) and Cusativin (CpN cleavage) and comparison with RNase T1 and RNase 4.
• Chromatography: Ion-pairing reversed-phase LC on ACQUITY Premier Oligonucleotide BEH C18 column with IonHance HFIP mobile phases.
• Mass Spectrometry: Xevo G3 QTof in negative ESI mode with accurate-mass acquisition.
• Data Analysis: waters_connect applications including mRNA Cleaver, MAP Sequence and Coverage Viewer for in silico digestion, product identification and sequence coverage visualization.

Main Results and Discussion

• Cleavage Specificity: MC1 cleaves at ApU, CpU and UpU sites; Cusativin at CpA, CpG, CpU and additional low-rate sites, providing complementary fragment sets.
• Reproducibility: Both RapiZyme enzymes showed consistent digestion profiles across batches with overlapping fragments that enhance mapping confidence.
• Sequence Coverage: Complete (100%) coverage of sgRNA achieved with both enzymes, compared to ~78% for RNase T1 and discrete non-overlapping maps for RNase 4.
• Fragment Characteristics: RapiZyme digests yield longer products with cyclic phosphates, reducing isomer ambiguity and strengthening MS signals.

Benefits and Practical Applications

• High Signal and Coverage: Unique overlapping fragments increase confidence and reduce ambiguity in sequence interpretation.
• Simplicity and Scalability: Single-pot, reproducible protocol suitable for automation in both research and QC settings.
• Streamlined Workflow: Integrated informatics accelerates data processing and supports regulatory compliance for therapeutic RNA characterization.

Future Trends and Potential Uses

As RNA therapeutics diversify into vaccines, gene editing tools and oligonucleotide drugs, advanced analytical solutions will be essential. The combination of complementary nucleases, high-resolution LC-MS and automated informatics will drive faster development cycles, improved quality control and support regulatory filings in industrial and clinical laboratories.

Conclusion

The use of RapiZyme MC1 and Cusativin together with optimized LC-MS conditions and waters_connect informatics enables reproducible, high-coverage mapping of therapeutic RNA. This workflow offers a robust platform for confirming sequence integrity and modifications in support of RNA drug development and quality control.

References

1. Gau B, Dawdy A, Wang HL et al. Oligonucleotide Mapping Via Mass Spectrometry to Enable Comprehensive Primary Structure Characterization of an mRNA Vaccine Against SARS-CoV-2. Sci Rep. 2023;13:9038.
2. 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(13):7021.
3. Gaye MM, Fox J, Vissers JPC, Reah I, Knowles C, Lauber M. Synthetic mRNA Oligo Mapping Using Ion Pairing Liquid Chromatography and Mass Spectrometry. Waters Application Note 720007669. 2022.