Combining Acoustic Force Spectroscopy and DNA Scaffold for High Throughput Measurement of Ligand-Receptor Kinetics at Single Molecule Resolution

Abstract

Single-molecule data are of great significance in biology, chemistry and medicine. However, experimental tools to accomplish high-throughput measurements are lacking. Acoustic force spectroscopy (AFS) is an emerging single-molecule technique which generates sound waves to apply force on a large population of microparticles in parallel. The force amplitude increases as the cube of the particle radius to reach 50-100 pN with a simple experimental set up. The tunability of the amplitude of the acoustic field allows dynamic control of the applied forces and makes the technique very suitable to study single molecules of interest tethered to microbeads and to the surface. We have exploited this configuration on a recently developed modular Junctured-DNA scaffold designed to study protein-protein interactions at the single-molecule level. A force calibration on individual molecules was performed simultaneously for the application of a cyclic step of force in order to produce repetitive binding-unbinding events on the very same ligand-receptor pairs. The unbinding kinetics under force was compared for the FRB-rapamycin-FKB12 complex previously investigated by magnetic tweezers at forces below 10 pN. We further characterize the force-extension response of Junctured-DNA beyond 10 pN. The method allows high-throughput measurements at the single-molecule level on a wide range of ligand-receptor interactions including nanobody-antigen and thus may provide novel information to characterize therapeutic molecules.