Insights into siRNA Duplex Stability from UV-Vis Concentration and Melting Temperature Analyses

Non-covalent RNA complexes such as siRNA duplexes are commercially established and are a continuously evolving class of oligonucleotide therapeutics, with many novel formats and modification patterns currently under development. The non-covalent nature of these duplexes merits unique considerations for development of analytical methods in comparison to single-stranded oligonucleotide therapeutics. Using UV-Vis absorbance measurements, thermal melting temperature analyses, and chromatography experiments, we explore the impact of siRNA concentration, solvent type and concentration, and salt type and concentration on siRNA duplex absorbance measurements and thermal melt data, and highlight the extent to which the hyperchromicity effect plays a role in these measurements, illuminating the stability of siRNA duplexes under various conditions. We also showcase the impact of the solution environment (i.e., the impact of the salt) during SEC of siRNA duplexes and single strands and the propensity for aggregation of single strands under certain chromatographic conditions. With this data, we present novel insights into the development of multiple critical quality attributes for RNA duplexes, including concentration, thermal melt analysis (Tm), and purity by SEC, and identify gaps and pitfalls in current industry practices. We share insights into unique considerations for the characterization of RNA duplexes guided by novel learnings from chromatography experiments as well as melting temperature (Tm) analysis and other spectroscopic technologies.