Digesta Ex Machina: Automating Sample Preparation for Oligo Mapping With Rapizyme™ MC1 and Cusativin on Andrew+™ Robot

Importance of Topic

RNA therapeutics have emerged as powerful tools in modern medicine, offering targeted interventions for previously incurable diseases. Comprehensive characterization of RNA quality attributes, including sequence integrity, purity, and modifications, is essential for ensuring safety and efficacy. Controlled enzymatic digestion followed by LC-MS analysis provides detailed oligonucleotide mapping, facilitating accurate sequence confirmation.

Goals and Overview

This study aimed to adapt a manual RNA digestion workflow using RapiZyme MC1 and Cusativin endonucleases to an automated platform based on the Andrew+ Pipetting Robot. Key objectives were to demonstrate equivalence between manual and automated sample preparations, to achieve high reproducibility across multiple samples, and to streamline throughput for routine quality control environments.

Methodology and Instrumentation

The protocol involved preparation of a 200 mM ammonium acetate digestion buffer, reconstitution of RapiZyme MC1 (pH 8.0) and Cusativin (pH 9.0) enzymes, and aliquoting of single guide RNA (sgRNA) targeting mouse GATA2. Digestions were performed at 30 °C for 15 minutes followed by heat inactivation at 70 °C or 75 °C. Samples were then transferred to screw-neck vials for direct LC-MS analysis.

Used Instrumentation

• Andrew+ Pipetting Robot with OneLab software
• Peltier+ connected device for incubation
• ACQUITY Premier UPLC system with Premier Oligonucleotide BEH C18 column
• Xevo G3 QTof mass spectrometer
• Thermomixer C and standard mechanical pipettes for manual comparison

Main Results and Discussion

Chromatograms obtained from manual and automated digestions of GATA2 sgRNA with both RapiZyme MC1 and Cusativin showed nearly identical profiles, with minor peak height variations attributed to thermal ramp rate differences. Automated processing of eight samples demonstrated excellent tube-to-tube reproducibility, confirmed by overlapping total UV chromatograms. Sequence coverage analysis using waters_connect MAP Sequence and Coverage Viewer applications achieved 100 percent confirmation for both enzyme digestions.

Benefits and Practical Applications

• Fully automatable workflow delivering eight digestions in under 45 minutes
• Reproducible and scalable sample preparation suitable for QC, analytical development, and research laboratories
• Direct compatibility with LC-MS analysis without additional cleanup steps
• Enhanced sequence coverage via overlapping oligonucleotide fragments

Future Trends and Possibilities

Automation of oligonucleotide mapping is expected to expand to larger RNA constructs such as mRNA vaccines and long non-coding RNAs. Integration with advanced informatics and artificial intelligence will streamline data interpretation and quality assessment. Miniaturized and high-throughput robotic platforms may further increase sample throughput and reduce reagent consumption.

Conclusion

The automated Andrew+ Pipetting Robot protocol for RapiZyme MC1 and Cusativin digestions delivers performance equivalent to manual methods while significantly improving throughput and reproducibility. This approach supports rigorous oligo mapping for RNA therapeutics characterization and quality control.

References

• Addepalli B Johnston T Reidy C Lauber M Tunable Digestions of RNA Using RapiZyme RNases to Confirm Sequence and Map Modifications Waters Application Note 720008539 September 2024
• Doneanu C Preston C Gorton M Johnson T Addepalli B Yu Y RNA Digestion Product Mapping Using an Integrated UPLC-MS and Informatics Workflow Waters Application Note 720008553 September 2024