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

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Summary

Acoustic force spectroscopy enables near-equilibrium measurements of streptavidin-biotin bond dissociation. This technique reconciles single-molecule force spectroscopy rates with bulk measurements, improving understanding of biomolecular interactions.

Keywords:
acoustic force spectroscopynear-equilibriumsingle-molecule force spectroscopystreptavidin−biotinunbinding

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Area of Science:

  • Biophysics
  • Biochemistry
  • Materials Science

Background:

  • Streptavidin-biotin (SA-b) bond dissociation is crucial in biological systems.
  • Existing single-molecule force spectroscopy (SMFS) methods yield dissociation rates (koff) inconsistent with bulk measurements due to non-equilibrium conditions.
  • Near-equilibrium SMFS is challenging due to requirements for high throughput, stability, and ultraslow loading rates.

Purpose of the Study:

  • To develop and apply *in situ* force calibration strategies for acoustic force spectroscopy (AFS).
  • To probe SA-b unbinding forces within the near-equilibrium regime.
  • To reconcile discrepancies in SA-b dissociation rates between SMFS and bulk methods.

Main Methods:

  • Development of *in situ* force calibration for acoustic force spectroscopy.
  • Application of AFS to measure SA-b unbinding forces at loading rates from 10 pN/s down to 10⁻³ pN/s.
  • Integration of new data with previous findings to cover 15 orders of magnitude in loading rate.

Main Results:

  • AFS successfully probed SA-b unbinding forces in the near-equilibrium regime.
  • Dissociation rates (koff) obtained via AFS align with bulk measurement values.
  • The study achieved a 15-order-of-magnitude dynamic range in loading rate for SMFS.

Conclusions:

  • Acoustic force spectroscopy provides a method to achieve near-equilibrium measurements for biomolecular bond dissociation.
  • The developed technique reconciles differing dissociation rates observed in SMFS and bulk assays.
  • This work expands the dynamic range of SMFS, offering a more comprehensive understanding of biomolecular energy landscapes.