Characterization and Sequencing of Duplex siRNA Using the BioAccord™ LC-MS System and Vion IMS QTof Mass Spectrometer in Combination With the waters_connect™ CONFIRM Sequence App

Significance of the Topic

Small interfering RNA (siRNA) has emerged as a transformative therapeutic modality in gene therapy and oncology. Ensuring precise sequence integrity, structural fidelity and high purity of duplex siRNA is critical to avoid off-target effects and maintain safety and efficacy standards. Advanced analytical workflows that integrate chromatographic separation, mass spectrometric detection and automated data processing are therefore essential for both research and regulated quality control applications.

Objectives and Study Overview

This work demonstrates a streamlined LC-MS strategy that exploits controlled column temperature and complementary mass spectrometric modes to:

• Maintain or denature duplex siRNA molecules on-column by adjusting temperature
• Evaluate duplex and single-strand purity and identity within a single run
• Obtain comprehensive sequence confirmation through automated informatics tools

Methodology and Instrumentation

Duplexes were prepared by annealing equimolar sense and antisense strands. Ion-pair reversed-phase chromatography (IP-RP) separations were performed at 25 °C (non-denaturing) and 60 °C (denaturing). Mass spectrometry modes included:

• Data-independent MSE fragmentation on the BioAccord LC-MS System, comprising an ACQUITY UPLC Premier I-Class PLUS, Tunable UV detector and RDa ESI-TOF detector
• Targeted MS/MS on a Vion IMS QTof instrument with precursor selection

Automated data analysis employed the waters_connect platform with the INTACT Mass App for deconvolution and purity assessment and the CONFIRM Sequence App for sequence mapping.

Main Results and Discussion

At 25 °C, the duplex remains intact on column but dissociates in the electrospray, allowing strand-specific deconvolution. The INTACT Mass App identified co-eluting species with high mass accuracy (< 10 ppm) and reported combined LC×MS purity (~90%). At 60 °C, on-column denaturation produced baseline separation of sense and antisense strands, eliminating chimeric fragment overlaps in MSE data. Under denaturing conditions, the CONFIRM Sequence App achieved 100% sequence coverage for both strands, whereas non-denaturing runs provided ~90% due to overlapping fragment ions. Targeted MS/MS on the Vion IMS QTof also delivered full sequence confirmation, underscoring the complementarity of data-independent and targeted approaches.

Benefits and Practical Applications

This dual-temperature LC-MS approach offers:

• Comprehensive assessment of duplex purity, identity and sequence in one analysis
• Automated, compliance-ready workflows accessible to non-expert users
• Seamless integration into development, manufacturing and quality control environments

Future Trends and Opportunities

Emerging advances in automation, high-resolution separations and ion mobility integration will further enhance oligonucleotide analysis throughput and accuracy. Expanded informatics capabilities, including machine-learning-driven spectral interpretation and real-time cloud reporting, will streamline regulatory compliance and accelerate therapeutic development across diverse oligonucleotide modalities.

Conclusion

The combined use of controlled column temperature, MSE fragmentation and targeted MS/MS provides a robust, automated solution for duplex siRNA characterization and sequencing. Leveraging the BioAccord and Vion IMS QTof platforms with waters_connect informatics enables precise purity measurements and full sequence confirmation within a streamlined, compliance-ready workflow.

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