Quantifying the force in flow-cell based single-molecule stretching experiments

Area of Science:

• Biophysics
• Single-molecule biophysics
• Biomolecular mechanics

Background:

• Flow-cell techniques are vital for studying DNA mechanics and protein-DNA interactions.
• Existing methods lack precise force characterization, often limited to <25 pN.
• Accurate force measurement is crucial for understanding molecular interactions.

Purpose of the Study:

• To quantitatively evaluate hydrodynamic drag in flow-cell based single-molecule manipulation.
• To establish a reliable method for force calibration in DNA stretching experiments.
• To extend the force measurement range in flow-cell assays.

Main Methods:

• Utilized a "tethered-bead" assay and a "bead-spring chain" model.
• Performed flow-cell based DNA pulling experiments.
• Calibrated force using bead's Brownian motion and DNA overstretching transition.

Main Results:

• Demonstrated a linear relationship between flow rate and total hydrodynamic force (0-110 pN).
• Developed a method to convert flow rate to force using a linear calibration factor.
• Calculated hydrodynamic forces and torques, showing good agreement between theoretical and measured force-extension curves.

Conclusions:

• Flow-cell assays provide precise single-molecule force measurements over a wide range (0-110 pN).
• This work offers critical insights into force characterization in flow-cell experiments.
• Establishes flow-cells as a versatile and cost-effective tool for biophysical studies.