Histopathological Assessment of IFI6- and RSAD2-DNA Aptamers in Oral Squamous Cell Carcinoma: A Preliminary Study...

Introduction: DNA aptamers are single-stranded nucleic acids capable of selectively binding target proteins and have emerged as potential alternatives to antibodies in molecular diagnostics. This study aimed to develop and characterize in silico designed DNA aptamers targeting Interferon alpha-inducible protein 6 (IFI6) and Radical S-adenosyl-L-methionine domain-containing protein 2 (RSAD2) for oral squamous cell carcinoma (OSCC). Methods: Genomic transfer RNA sequences from Homo sapiens, Mus musculus, and Escherichia coli were computationally truncated and optimized to generate DNA aptamer candidates ranging from 35-50 mers. Secondary and tertiary structures were generated using Mfold and RNAComposer, followed by molecular docking with AutoDock Vina and molecular dynamics simulations in GROMACS to evaluate docking interactions and structural behavior comparatively. Selected DNA aptamer candidates were synthesized and evaluated by aptahistochemistry (AHC) to qualitatively explore preliminary tissue reactivity in formalin-fixed paraffin-embedded OSCC tissues under optimized conditions. Results: Molecular docking demonstrated favorable comparative docking scores ranging from -15.6 to -18.7 kcal/mol. RMSD analysis demonstrated that selected IFI6 and RSAD2 DNA aptamer-protein complexes reached a plateau with relatively small fluctuations following maximum RMSD values. In contrast, RMSF analysis identified increased flexibility predominantly within loop regions. Cross-reactivity analysis explored preliminary target selectivity, with no interactions observed within the selected target-binding regions. During AHC optimization, the 50-IFI6 and 45-RSAD2 DNA aptamer candidates exhibited cytoplasmic brown granular staining in >50% of OSCC tumor cells under the optimized experimental conditions. Conclusions: The findings support the applicability of an integrated in silico workflow for generating structurally optimized DNA aptamer candidates and provide preliminary proof-of-concept for their exploratory histopathological application in OSCC.