Revealing New Analytical Insights into RNA Complexes: Divalent siRNA Characterization by Liquid Chromatography and Mass Spectrometry.

Accurate characterization of therapeutic RNA, including purity and identity, is critical in drug discovery and development. Here, we utilize denaturing and non-denaturing chromatography for the analysis of ∼25 kDa divalent small interfering RNA (di-siRNA), which comprises a complex 2:1 triplex structure. Ion pair reversed-phase (IPRP) liquid chromatography (LC) experiments with UV absorbance and mass spectrometry (MS) showcase a single denaturing LC method for identity confirmation, impurity profiling, and sequencing with automated MS data interpretation. IPRP, size exclusion chromatography (SEC), and melting temperature () experiments showcase the need for consideration of chromatographic conditions in evaluating noncovalent siRNA structures─here, low-temperature IPRP experiments (generally considered "non-denaturing") indicate denaturation of the noncovalent complex for certain di-siRNA sequences, while SEC data indicate that di-siRNA aggregation can be vastly underestimated due to sample dilution prior to LC experiments. Furthermore, SEC data critically show the propensity of denatured di-siRNA samples to renature under SEC mobile phase conditions upon exposure to high ionic/salt solutions, corroborated by experiments. This work highlights the need for consideration of the noncovalent nature of certain RNA therapeutics during sample preparation, method development, and analytical characterization and the often sequence-specific strength of complex formation, which may significantly affect analytical results obtained for such molecules. Attention to the highly dynamic nature of the duplex RNA structure, which is heavily influenced by its environment, must be considered during analytical method development.