Oligo Mapping of sgRNA Digests: Leveraging Xevo MRT Mass Spectrometer Performance and Streamlining Data Analysis

Significance of the Topic

Therapeutic RNAs such as single-guide RNAs (sgRNAs) are central to precision gene editing and emerging diagnostic and therapeutic applications. High-resolution mapping of digestion products confirms sequence integrity and modification patterns, ensuring functionality and regulatory compliance. Advances in LC-MS instrumentation and bioinformatics streamline sgRNA characterization, improving confidence in analytical workflows.

Objectives and Overview

This study demonstrates a compliance-ready workflow for complete oligo mapping of RapiZyme MC1-digested sgRNA using the Xevo MRT Mass Spectrometer and automated data analysis. Key goals include:

• Achieving 100% unique sequence coverage of a 100-mer sgRNA standard.
• Leveraging sub-ppm mass accuracy and high resolution to confidently assign digestion products.
• Integrating elevated energy fragmentation to resolve isomeric oligonucleotides.

Methodology and Instrumentation

Sample Preparation:

• Waters 100-mer sgRNA standard denatured and digested with RapiZyme MC1 for 30 min at 30 °C.
• Digests injected (5 µL) onto UPLC for direct LC-MS analysis.

Chromatography and Mass Spectrometry:

• ACQUITY Premier UPLC with Oligonucleotide BEH C18 column, 70 °C, 400 µL/min flow.
• Xevo MRT QTOF Mass Spectrometer operated in ESI(–) MSE mode at 2 Hz, 550 °C desolvation, sub-ppm mass accuracy, ~100 000 resolution.

Informatics Workflow:

• mRNA Cleaver MicroApp for in silico digest prediction.
• MAP Sequence App for automated MS1 matching and sequence coverage visualization.
• Coverage Viewer MicroApp to display coverage map.
• CONFIRM Sequence App to assign elevated energy fragments and resolve sequence isomers.

Key Results and Discussion

Full unique sequence coverage (100%) was obtained through overlapping digestion products and predictable missed cleavages by RapiZyme MC1. All predicted oligonucleotide masses matched experimental MS1 data within sub-ppm accuracy. The Xevo MRT’s high resolution resolved multiple charge states and isotope patterns, exemplified by an 8-mer product at 100 000 resolution. Two structural isomers (7-mers) were chromatographically separated and unambiguously assigned via elevated energy MSE fragmentation using the CONFIRM Sequence App, achieving complete MS2 coverage.

Benefits and Practical Applications

• Rapid, automated sgRNA sequence verification ensures quality control in research and therapeutic development.
• Compliance-ready informatics within waters_connect platform supports regulatory documentation.
• Enhanced cleavage specificity of RapiZyme MC1 improves coverage over traditional RNases.
• High sensitivity and speed of Xevo MRT enable routine high-throughput analysis.

Future Trends and Applications

Integration of real-time data analysis and further automation can accelerate RNA therapeutic development. Emerging ribonucleases with tailored specificity may provide deeper insight into RNA modifications. Coupling high-resolution instruments with AI-driven data interpretation will further simplify oligo mapping and broaden applications in synthetic biology, diagnostics, and personalized medicine.

Conclusion

The demonstrated workflow combining RapiZyme MC1 digestion, Xevo MRT high-resolution MS, and waters_connect informatics delivers comprehensive, accurate sgRNA characterization. Sub-ppm mass accuracy and automated sequence assignment enable confident mapping of digestion products, including isomer differentiation, supporting rigorous quality control in RNA research and therapeutic pipelines.

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