Top-Down Characterization of Complete siRNA Complexes by Direct Infusion Nanospray-MSⁿ

Area of Science:

• Mass Spectrometry
• Biochemistry
• Pharmaceutical Analysis

Background:

• Small interfering RNA (siRNA) therapeutics require comprehensive characterization.
• Existing mass spectrometry methods face challenges with complex siRNA structures and modifications.
• Top-down analysis offers a direct approach to characterize intact biotherapeutics.

Purpose of the Study:

• To evaluate top-down mass spectrometry strategies for characterizing complete siRNA complexes.
• To develop methods for analyzing siRNA duplexes and stem-loop structures with N-acetylgalactosamine (GalNAc) modifications.
• To enable sequence and modification site determination without prior hydrolysis or separation.

Main Methods:

• Direct gas-phase activation of intact siRNA complexes.
• Collision-induced dissociation (CID) and in-source activation for fragmentation.
• Use of denaturing agents to destabilize complex structures.
• MS/MS analysis of dissociated strands and backbone fragments.

Main Results:

• Standard CID fragmented GalNAc and nucleobases, hindering sequence analysis.
• In-source activation enabled full sequencing of simple duplexes but not stem-loops.
• Denaturing agents successfully induced destabilization and revealed informative backbone fragments.
• Combined strategies allowed full characterization of siRNA sequences, GalNAc sites, and structures.

Conclusions:

• Top-down mass spectrometry can comprehensively characterize pharmaceutical siRNA forms.
• Strategy selection must be adapted to specific siRNA morphologies (duplex vs. stem-loop).
• This minimalistic approach reduces sample consumption and analysis time, aiding biotherapeutic development.