Aptamer binding assays and molecular interaction studies using fluorescence anisotropy - A review

Abstract

Binding of nucleic acid aptamers to specific targets and detection with fluorescence anisotropy (FA) or fluorescence polarization (FP) take advantage of the complementary features of aptamers and the fluorescence techniques. We review recent advances in affinity binding assays using aptamers and FA/FP, with an emphasis on studies of molecular interactions and identification of binding sites. Aptamers provide several benefits, including the ease of labelling fluorophores on specific sites, binding-induced changes in aptamer structures, hybridization of the aptamers to complementary sequences, changes in molecular volume upon binding of the aptamer to its target, and adsorption of aptamers onto nanomaterials. Some of these benefits have been utilized for FA/FP assays. Once the aptamer binds to its target, the resulting changes in molecular volume (size), structure, local rotation of the fluorophore, and/or the fluorescence lifetime influence changes to the FA/FP values. Measurements of these fluorescence anisotropy/polarization changes have provided insights into the molecular interactions, such as the binding affinity and the site of binding. Studies of molecular interactions conducted in homogeneous solutions, as well as those with separations, e.g., capillary electrophoresis, have been summarized in this review. Studies on mapping the position of binding in aptamers at the single nucleotide level have demonstrated a unique benefit of the FA/FP techniques and pointed to an exciting direction for future research.

Keywords: Affinity interaction; Aptamer binding; Capillary electrophoresis; Characterization of binding sites; Fluorescence anisotropy; Fluorescence polarization; laser-induced fluorescence; proteins.