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 (<i>k</i>_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 <i>in situ</i> 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 <i>k</i>_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.