Electrostatically driven binding of a C ₆₀ -conjugated AT11 aptamer to nucleolin: molecular dynamics and free energy calculations

Photodynamic therapy (PDT) faces a significant challenge in achieving selective targeting of cancer cells. To address this, we investigated the conjugation of the C60 fullerene, a potent photosensitizer, with the AT11 DNA aptamer, a thermostable G-quadruplex known to target the overexpressed nucleolin (NCL) protein in cancer cells. Using molecular docking, molecular dynamics (MD) simulations, and free energy calculations with the accelerated weight histogram (AWH) method, we characterised the binding mechanism of both the unmodified AT11 aptamer and its C60-Thr2 conjugate to the RBD1,2 domains of NCL. Our results reveal a fit-induced recognition mechanism where NCL undergoes a conformational change, folding its two domains around the aptamer. Binding is primarily stabilised by electrostatic interactions between lysine residues of NCL and phosphate backbone of the AT11 loop. Conjugation of the C60-Thr2 to the 3’ end of the aptamer caused only a minor perturbation to this binding mechanism and a slight reduction in binding free energy (from ΔG = 12.45 ± 5 kcal/mol to ΔG = 10.56 ± 5 kcal/mol), indicating that the aptamer's targeting capability is largely preserved. These findings suggest that the AT11-C60-Thr2 conjugate is a highly promising candidate for targeted PDT, combining efficient photosensitizer delivery with maintained high binding affinity to nucleolin.