Binding Characterization Of Small Protein-Conjugated ssDNA Aptamer To Recombinant Human ICAM-1

This study investigates the potential of a protein-DNA aptamer conjugate to enhance aptamer binding to recombinant human intercellular adhesion molecule 1 (rhICAM-1). Aptamers are single-stranded nucleic acids that bind target molecules through hydrogen bonding and hydrophobic interactions. Conjugating aptamers with antibodies or proteins has been shown to improve their binding affinity. Using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), eight rounds of selection were performed with ICAM-1-coupled Dynabeads Protein A, identifying a G/C-rich aptamer, DI05, as having the strongest binding affinity to rhICAM-1. An antibody inhibition assay demonstrated a significant reduction in rhICAM-1 binding to immobilized ap-tamers (DI05, DI20, DI31, and DI33). Additionally, the binding affinity of eGFP-conjugated DI05 to rhICAM-1 was higher than that of unconjugated DI05. Docking simulations revealed close contact between DI05 and ICAM-1, with interactions primarily mediated by hydrogen bonds within three hairpin structures at ≤2.8 Å. These findings highlight the potential of aptamer-small protein conjugates as a promising strategy to enhance aptamer binding characteristics.