SEQUENCE CONFIRMATION OF OLIGONUCLEOTIDES VIA AUTOMATED TOP-DOWN SPECTRAL ANNOTATION

Significance of Topic

Resurgent growth in oligonucleotide therapeutics and regulatory demands drive the need for efficient sequence confirmation workflows. Top down mass spectrometry offers full length sequence verification for both modified and unmodified oligonucleotides while supporting complex modifications.

Study Objectives and Overview

This work describes three analytical workflows and a new software tool called Spectrum for automated top down spectral annotation. A mixture of twenty base oligonucleotides including standard RNA, 2 prime O methyl modified RNA, phosphorothioate backbone RNA and DNA were analyzed to assess sequence coverage and mass accuracy.

Methodology and Instrumentation

• Sample Preparation: Oligonucleotides reconstituted in RNase free water and combined at 10 micromolar equimolar concentration
• Chromatography: Waters UPLC I class system with BEH C18 column at 60 C and gradient based on triethylamine HFIP buffers in water and methanol
• Mass Spectrometry: Waters Xevo G2 XS QTof in negative electrospray mode; acquisition range m/z 500 to 3000
• Acquisition Workflows: full MS scan; data dependent MS MS selecting top four precursors; data independent acquisition MSE with alternating low and high collision energy scans
• Data Processing: MassLynx and Spectrum software automatically deconvolute spectra and annotate fragment ions for coverage and mass error calculations

Key Results and Discussion

• Sequence coverage between eighty and ninety percent achieved across charge states from 3 to 9
• Higher charge states yielded richer fragmentation at lower collision energies
• Data dependent MS MS provided improved coverage for modified RNA compared to MSE
• Spectrum tool delivered automated fragment assignment with mass error below eight parts per million

Benefits and Practical Applications

This automated annotation workflow reduces manual effort, accelerates method development and supports quality control of therapeutic oligonucleotides with diverse modifications.

Future Trends and Potential Applications

Further enhancements may include support for additional chemical modifications, real time data processing, integration with laboratory information management systems and application to more complex sequence mixtures or large scale biomolecules.

Conclusion

The integration of top down mass spectrometry workflows with automated Spectrum annotation provides a robust and efficient platform for comprehensive sequence confirmation of oligonucleotides, meeting the needs of research development and regulatory compliance.