Thermodynamic and Structural Consequences of Nucleotide Deletion in DNA/RNA Heteroduplexes

Abstract Nucleotide deletions in nucleic acid duplexes can induce noncanonical structures such as bulges and terminal fraying, which may influence the off-target effects of antisense oligonucleotides (ASOs). However, the thermodynamic and structural properties of deletion-containing DNA/RNA heteroduplexes remain poorly understood. In this study, we systematically investigated the effects of single-nucleotide deletions at different positions in a twenty-mer DNA strand hybridized with complementary RNA. Thermodynamic parameters were determined from UV melting analyses, and structural changes were examined using NMR imino proton spectroscopy. All deletion-containing duplexes exhibited reduced thermodynamic stability relative to that of the fully matched duplex. The extent of destabilization strongly depended on the deletion position: terminal deletions caused only modest destabilization, whereas deletions near the central region produced substantially larger energetic penalties. The largest difference in stability among the duplexes corresponded to an approximately 103-fold difference in binding constants. These results suggest a structural transition between terminal fraying and internal bulge formation. Central deletions likely induce structural strain and disruption of base-stacking interactions, whereas terminal deletions can be accommodated through fraying at the helix ends. NMR analyses further revealed that structural perturbations caused by nucleotide deletion were not always localized to the deletion site. In several duplexes, disappearance of imino proton signals was observed at positions a few nucleotides away from the deletion, indicating long-range propagation of structural instability and reduced helical cooperativity. These findings provide new insights into the structural stability of DNA/RNA heteroduplexes and provide a fundamental physicochemical framework for understanding deletion-containing DNA/RNA heteroduplexes and for evaluating their potential relevance to off-target interactions in ASO systems.