RNA Digestion Product Mapping Using an Integrated UPLC-MS and Informatics Workflow

Importance of the Topic

Single-guide RNAs (sgRNAs) are central to CRISPR-Cas9 gene editing and therapeutic development. Accurate sequence confirmation of sgRNAs and modified oligonucleotides supports safety, efficacy, and regulatory compliance. UPLC-MS combined with advanced informatics addresses the need for fast, reliable mapping of RNA digestion products, accelerating research, quality control, and drug development.

Objectives and Study Overview

This study presents an integrated workflow for enzymatic digestion product mapping of sgRNAs using UPLC-MS and automated data processing. Key goals include developing informatics tools for rapid assignment of predicted and experimental digest products, evaluating multiple ribonucleases to enhance sequence coverage, and demonstrating compliance-ready analytics on the waters_connect platform.

Methodology and Instrumentation

Sample Preparation and Enzymes:

• 100-mer HPRT1 sgRNA with 2’-OMe modified and phosphorothioate linkers at both termini.
• Enzymes: RNase T1, hRNase4, RapiZyme MC1 (RNase T2), and RapiZyme Cusativin.

UPLC-MS Analysis:

• System: ACQUITY Premier UPLC with Xevo G3 QTof MS.
• Column: BEH C18 oligonucleotide, 2.1×150 mm, 1.7 µm.
• Mobile phase: 10 mM DPEA / 40 mM HFIP in water (A) and 50% methanol (B), IP-RP gradient at 60°C.

Informatics Tools:

• UNIFI Scientific Information System 3.6.
• mRNA Cleaver MicroApp for in-silico digest prediction.
• MAP Sequence App for automated mass matching.
• Coverage Viewer for sequence coverage visualization.
• CONFIRM Sequence App for fragment confirmation.

Key Results and Discussion

Workflow Implementation:

• mRNA Cleaver predicted oligo products for each enzyme considering cleavage specificity and missed cleavages.
• MAP Sequence matched predicted masses to experimental data in under 3 minutes per dataset.
• Coverage Viewer summarized unique sequence coverage per enzyme.

Enzyme Performance:

• RNase T1: 22 predicted products, ~95% coverage, some identical sequence ambiguities due to G-specific cleavage.
• hRNase4: 14 products with U_A/U_G specificity, ~90% coverage, minor isomer assignments.
• RapiZyme MC1: multiple cleavage sites including modified U, achieved 100% coverage on the 100-mer substrate.
• RapiZyme Cusativin: cytidine-specific cleavage, complementary coverage, confirmed unique Y_Y motif cleavage for modified residues.

Combined Approach:
Integrating results from all four enzymes maximized overall sequence confirmation and confidence.

Benefits and Practical Applications

The compliance-ready waters_connect workflow offers rapid, automated RNA mapping suitable for research, development, and quality control in RNA therapeutics and diagnostics.

Future Trends and Opportunities

Expansion of enzyme panels with tailored specificities, deeper integration of fragment confirmation, application to larger mRNA constructs, and deployment in manufacturing QC and personalized medicine are anticipated.

Conclusion

The integrated UPLC-MS and informatics workflow with multiple ribonucleases significantly enhances sgRNA sequence verification, delivering high coverage, streamlined data analysis, and robust analytical support for RNA-based drug development.

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