Structural and Thermodynamic Insights for Enhanced SNP Detection Using <i>N</i> -Benzimidazole Oligonucleotides

Oligodeoxyribonucleotides bearing <i>N</i>-benzoazole modifications in the phosphate group (PABAO) represent a recently developed class of nucleic acid derivatives with potential in various applications, in particular for SNP detection. This study investigates the physicochemical and substrate properties of DNA/DNA and PABAO/DNA complexes, both perfectly matched and containing a single mismatch near the 3'-end. The <i>N</i>-benzimidazole modification enhances mismatch discrimination during hybridization in high ionic strength buffers, while thermal destabilization upon mismatch introduction remains similar for both complex types under PCR-like conditions. Structural studies revealed local perturbations induced by the modification and/or mismatch. Primer elongation efficiency by <i>Taq</i> DNA polymerase was significantly reduced in PABAO-containing complexes depending on the nucleotide composition, the presence and type of mismatch, and the position of the modification. The yield of full-length extension products increased when modifications were positioned farther from the 3'-end of the primer in mismatched duplexes, with no strict correlation to mismatch type or flanking bases. Modifications at the first internucleotide phosphate position disrupt proper primer alignment within the catalytic center of <i>Taq</i> DNA polymerase, impairing its enzymatic function. The predominant byproducts corresponded to oligomers containing modifications at the fourth internucleotide phosphate of the primer. Molecular dynamics simulations demonstrated the stereospecific binding of the <i>Rp</i> isomer of the <i>N</i>-benzimidazole moiety to a hydrophobic pocket in the thumb domain of the enzyme that underlies the observed effect. We found that PABAOs enable mismatch discrimination while maintaining the highest yield of full-length elongation products for studied complementary primers, typically through modifications at the third internucleotide phosphate from the primer's 3'-end. This work establishes a physicochemical basis for leveraging PABAOs as probes for SNP detection in molecular diagnostics, particularly PCR-based applications, emphasizing the critical role of modification position and mismatch type in balancing specificity and efficiency.