Comprehensive oligonucleotide analysis

Importance of the topic

Oligonucleotide based therapeutics and mRNA products demand precise analytical workflows to guarantee quality, efficacy and safety. High resolution mass spectrometry coupled with advanced data processing addresses challenges such as multiple chemical modifications and complex fragmentation patterns, supporting discovery, development and quality control in nucleic acid based biotherapeutics.

Study objectives and overview

This work illustrates a comprehensive analytical strategy that unifies intact mass analysis, sequence specific isotopic modeling, fragmentation driven sequence confirmation and mRNA mapping. The aim is to deliver accurate identification, relative quantitation and confident sequence validation for heavily modified oligonucleotides and long mRNA constructs.

Methodology and instrumentation

Key elements of the workflow include:

• Intact mass deconvolution using a sliding window algorithm combined with sulfur isotope abundance models to resolve phosphorothioate modifications
• Fragmentation prediction by a kinetic model algorithm to generate MS2 spectra and intensity profiles for sequence confirmation
• In silico RNase digestion for mRNA mapping, followed by interactive coverage map visualization and confidence scoring

Main results and discussion

Intact mass analysis achieved high mass accuracy and resolved fully phosphorothioated oligonucleotides along with related impurities. Fragmentation data enabled confident differentiation of isomeric species and confirmed base order beyond full scan limitations. mRNA mapping yielded extensive sequence coverage through automated RNase digestion, fragment matching and custom filter application, supported by a randomized sequence generator to assess false positives.

Benefits and practical applications

The integrated approach facilitates rapid and reliable characterization of oligonucleotide therapeutics, including detection of expected and unexpected modifications, impurity profiling and relative quantitation. The mRNA mapping workflow supports vaccine and gene therapy development by delivering detailed sequence coverage and statistical confidence metrics.

Future trends and opportunities

Emerging directions include incorporation of machine learning for improved fragmentation prediction, expansion of support for diverse chemical modifications, and real time data processing to accelerate high throughput analyses. Advances in automation and spectral modeling will continue to enhance nucleic acid analysis in both research and quality control environments.

Conclusion

This unified platform combines high resolution mass spectrometry with advanced software capabilities to provide a comprehensive solution for oligonucleotide and mRNA characterization, ensuring accurate, high confidence analysis of complex nucleic acid therapeutics.

Instrumentation used

• Thermo Scientific Orbitrap mass spectrometer
• BioPharma Finder software