Near-Equilibrium Unbinding of Streptavidin-Biotin Using Single-Molecule Acoustic Force Spectroscopy

The dissociation of the streptavidin-biotin (SA-b) bond has been widely characterized using bulk and single-molecule force spectroscopy (SMFS) techniques. However, dissociation rates (k_off) from SMFS (∼10^-1 s^-1) differ from bulk approaches (∼10^-6-10^-5 s^-1), likely because SMFS measurements are conducted far from equilibrium. Near-equilibrium SMFS requires high-throughput measurements to obtain large enough statistics and high stability over long periods for ultraslow loading force rates measurements, impractical in most SMFS techniques. Here, we developed in situ force calibration strategies for acoustic force spectroscopy to probe SA-b unbinding forces in the near-equilibrium regime, from 10 down to 10^-3 pN/s. The resulting k_off matches bulk measurements values. Combined with our previous data, we covered 15 orders of magnitude in loading rate, expanding the dynamic range of SMFS and enabling a more complete description of the energy landscape of biomolecular processes.