Two Interconvertible Folds Modulate the Activity of a DNA Aptamer Against Transferrin Receptor

Thanks to their ability to recognize biomolecular targets with high affinity and specificity, nucleic acid aptamers are increasingly investigated as diagnostic and therapeutic tools, particularly when their targets are cell-surface receptors. Here, we investigate the relationship between the folding of an anti-mouse transferrin receptor DNA aptamer and its interaction with the transferrin receptor both in vitro and in living cells. We identified and purified two aptamer conformers by means of chromatographic techniques. Fluorescence-anisotropy measurements showed that only one fold is able to bind mouse transferrin receptor. Besides displaying enhanced endocytosis in living mouse fibroblasts, the purified active fold is internalized also in human pancreatic cancer cells. Starting from these observations, we rationally designed variations of the parent sequence aimed at stabilizing the active fold, and consequently increase aptamer activity. A truncated version and full-length mutants with higher affinity than the parent sequence are shown.