Investigating the Strength of the Sense and Antisense Oligonucleotide Interaction to Form siRNA Duplexes Using the BioAccord™ LC-MS System

Importance of the topic

The analysis of intact small interfering RNA (siRNA) duplexes is central to development, quality control, and stability testing of oligonucleotide therapeutics. Methods that preserve duplex higher-order structure during separation and ionization enable direct assessment of strand association, identity, and impurities without relying on denaturing conditions. The study demonstrates a HILIC-UV/MS workflow on a BioAccord ESI-TOF platform that supports native-like mass analysis of intact siRNA duplexes while avoiding toxic ion-pairing reagents, offering practical advantages for analytical labs and regulated environments.

Objectives and overview of the study

The primary aim was to develop and demonstrate a single HILIC-UV/MS assay capable of:

• Separating and detecting intact siRNA duplexes under native mass spectrometry conditions.
• Measuring neutral monoisotopic masses of intact duplexes using BayesSpray deconvolution.
• Comparing the gas-phase stability of different duplexes to infer relative strand interaction strength (sense–antisense binding).

The work analyzed three commercially sourced siRNA duplexes (SEM1, AIDA, ATPase PAAT) to show method robustness across different sequences and impurity profiles.

Methodology

Sample preparation and chromatographic approach:

• Lyophilized duplexes were reconstituted to 50 µM stock and diluted 10-fold into a HILIC-compatible solvent (final 5 µM) prior to injection (1 µL).
• Mobile phases were ammonium acetate–based and MS-friendly, avoiding HFIP and ion-pairing reagents.
• HILIC separation conditions preserved duplex integrity (column held at 35 °C, below expected duplex Tm).

Key mass spectrometry strategy:

• Electrospray ionization in negative mode (ESI-) under optimized source conditions to minimize in-source dissociation.
• Acquisition on a time-of-flight (TOF) analyzer with a mass range of 400–5000 m/z; deconvolution performed with BayesSpray in the INTACT Mass App for neutral monoisotopic mass determination.

Used instrumentation

The principal instrumentation and conditions summarized from the study:

• LC system: ACQUITY Premier UPLC with ACQUITY Premier BEH Amide HILIC column (1.7 µm, 130 Å, 2.1 × 50 mm).
• MS: BioAccord LC-MS system with ESI-TOF detector, negative ion mode.
• UV detection: ACQUITY UPLC TUV at 260 nm.
• Representative source parameters: capillary voltage ~0.8 kV, cone voltage 20 V, desolvation temperature 500 °C, desolvation gas (N2) pressure 6.5 bar.
• Mobile phases: Solvent A = 75% acetonitrile, 10 mM ammonium acetate, pH ~6.8; Solvent B = 25% acetonitrile in water, 10 mM ammonium acetate, pH ~6.8.

Main results and discussion

HILIC performance and intact duplex detection:

• Each siRNA produced a single dominant HILIC UV peak at 35 °C, indicating intact duplex retention despite sequence differences and co‑existing impurities from limited strand purification.
• HILIC provided strong retention and separation of intact duplexes from single-strand impurities while using MS-compatible mobile phases.

Native MS findings and assessment of duplex stability:

• ESI-MS spectra of HILIC-separated duplexes displayed a narrow charge-state distribution (predominantly −5 to −7), consistent with compact, native-like duplex structures in the gas phase.
• Ion-pair reversed-phase (IP-RP) conditions (same temperature) led to complete duplex denaturation and detection of single strands only, illustrating HILIC advantage for native analysis.
• Two duplexes (SEM1 and AIDA) showed partial in-source dissociation to single strands despite optimized source settings; the ATPase PAAT duplex exhibited minimal dissociation and markedly greater gas-phase stability.
• Because identical ESI source parameters were used across samples, differences in in-source dissociation are attributed to intrinsic differences in sense–antisense interaction strength (sequence-dependent thermodynamic stability), rather than instrumental variability.

Data processing:

• BayesSpray deconvolution in the INTACT Mass App yielded neutral monoisotopic mass assignments for intact duplexes, supporting identity confirmation and mass-based purity assessment.

Benefits and practical applications

Key advantages of the described HILIC-UV/MS workflow:

• Preserves siRNA duplex higher-order structure enabling direct intact-mass analysis and assessment of strand association strength.
• Avoids toxic HFIP and ion-pairing reagents, using MS-friendly ammonium acetate buffers compatible with TOF detection and downstream workflows.
• Enables simultaneous UV and MS data capture for qualitative (identity, duplex vs strands) and quantitative (relative abundance, impurity profiling) assessments.
• Supports method development, stability studies, identity confirmation, and potential QC workflows within an integrated data environment.

Future trends and applications

Potential developments and uses building on this work:

• Extension to larger or chemically modified oligonucleotides, including assessment of duplexes with 2’-modifications, conjugates, or long guide RNAs.
• Integration of ion mobility spectrometry (IMS) to resolve conformers and better characterize duplex topology and gas-phase compactness.
• Automated deconvolution and reporting workflows for regulated environments to facilitate QC and batch release testing.
• Use of quantitative native MS approaches to estimate duplex association/dissociation kinetics and relative binding strengths across sequence variants.
• Broader adoption of HILIC native-MS methods in regulatory submissions as orthogonal characterization supporting identity and stability claims.

Conclusion

This application study demonstrates that HILIC-UV/MS on a BioAccord ESI-TOF system can preserve and detect intact siRNA duplexes, enabling direct mass confirmation and providing a means to compare relative sense–antisense interaction strengths via observed in-source dissociation behavior. The method offers practical advantages—strong retention of polar oligonucleotides, separation from single-strand impurities, MS-friendly mobile phases, and integrated data processing—that make it a valuable approach for characterization and stability testing of siRNA therapeutics.

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