Exploring Molecular Binding: A Fluorescence Anisotropy Lab for Undergraduates

Abstract

Measuring interactions between macromolecules is essential for elucidating their dynamics in solution and is critical for the design and study of potential therapeutics. Fluorescence anisotropy has been a powerful and widely used tool for studying binding interactions. When a fluorescent partner is excited with polarized light while bound to its cognate ligand, the emitted light is partially polarized. This process is dependent on the concentration of bound molecules, permitting the determination of the binding affinity. Here, we outline a highly modular undergraduate-level laboratory in which students use fluorescence anisotropy to measure binding interactions between a synthetic nucleic acid aptamer and its protein target. Students learn the theory of functional nucleic acids and the principles of fluorescence while exploring the relevance of aptamer sequence/structure activity relationships via mutations to aptamers. With this approach, students can deepen their knowledge about macromolecular interactions and are able to develop valuable analytical and biophysical laboratory skills. This experiment is highly adaptable to suit a range of funding and instructor availability, making it accessible and tailorable to most laboratory settings.