Native Measurement of Aptamer Folding and Binding Energies Using a Free Energy Shift Assay

Folding and binding energies (ΔG _fold ^° and ΔG _bind ^°) are critical thermodynamic parameters for characterizing the stability and functionality of aptamers. However, it remains difficult to directly characterize aptamer folding and binding in the native environment. Here, we describe a free energy shift assay (FESA) as a simple and instrument-free method to characterize the folding and binding thermodynamics of aptamers. FESA leverages the thermodynamic impact of the aptamer on a panel of toehold-mediated exchange reactions with well-characterized energetics to determine its folding and binding free energies. Using this principle, we successfully determined the ΔG _fold ^° and ΔG _bind ^° of a classic aptamer, thrombin binding aptamer (TBA), against varying cations, molecular crowding, and a cell-mimicking environment. Because our method is a molecular tool that can be easily adapted in any biochemistry laboratory without the need for specialized instruments, we anticipate that it will find broad applications in the thermodynamic characterization of aptamers and other functional or noncanonical nucleic acids.