Surface Plasmon Resonance Analysis for Evaluating ASO Targeting Structured RNA

Antisense oligonucleotides (ASOs) are nucleic acid therapeutics that regulate gene expression through sequence-specific hybridization with target RNA. Under physiological conditions, many target RNAs adopt higher-order structures, which can strongly influence ASO accessibility and binding behavior. Although UV melting analysis is widely used to evaluate the thermal stability of ASO/RNA duplexes, this approach does not adequately account for the structural features of target RNAs. In this study, we investigated the utility of surface plasmon resonance (SPR) analysis as an in vitro method to evaluate ASO binding while considering RNA structural constraints. Multiple ASOs were designed to target PRF84, an 84-nucleotide RNA motif that induces -1 programmed ribosomal frameshifting in HIV-1 <i>gag</i>-<i>pol</i> expression. SPR analyses were performed to compare ASO interactions with complementary RNA fragments and with structurally folded PRF84. The results demonstrated that identical ASOs exhibited distinct binding behaviors depending on whether the target was a complementary RNA or PRF84, indicating that RNA structure significantly affects ASO binding. These findings suggest that SPR analysis enables the evaluation of ASO-RNA interactions taking structure into account, and may be a useful alternative approach to conventional UV melting analysis-based ASO screening.