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, the dissociation rates ( k _off ) from SMFS (∼10 ⁻¹ s ^-1 ) typically do not align with those 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 time measurements at ultraslow loading force rates, impractical in most SMFS techniques. Here, we developed in-situ force calibration strategies for acoustic force spectroscopy (AFS) to probe the unbinding forces of SA-b in the near equilibrium regime, from 10 pN/s down to 10 ^{− 3} pN/s. The resulting k _off was in excellent agreement with that from bulk measurements on the very same system. Combined with our previous SA-b data, we covered 15 orders of magnitude in loading rate, setting the ground for SMFS over the widest dynamic range, essential to fully describe the energy landscape of biomolecular processes.