Force feedback effects on single molecule hopping and pulling experiments

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Single-molecule optical tweezers are crucial for studying biological systems like nucleic acids.
• Force unzipping experiments analyze folding/unfolding thermodynamics and kinetics.
• Hopping experiments measure kinetic rates using different optical tweezer modes.

Purpose of the Study:

• To compare hopping and pulling experiments in constant force mode (CFM) and passive mode (PM) for DNA hairpin kinetics.
• To evaluate the accuracy of CFM and PM hopping experiments at varying temperatures (6°C, 25°C, 45°C).
• To assess the reliability of CFM and PM pulling experiments for kinetic rate determination.

Main Methods:

• Performed single-molecule hopping and pulling experiments on a DNA hairpin.
• Utilized optical tweezers in both constant force mode (CFM) with feedback and passive mode (PM) without feedback.
• Conducted experiments at three distinct temperatures to capture a range of kinetic rates.

Main Results:

• Equilibrium hopping experiments in CFM and PM accurately measured kinetics at 6°C (slow kinetics).
• CFM hopping experiments overestimated molecular parameters at 45°C (fast kinetics) due to feedback limitations.
• Non-equilibrium pulling experiments in both CFM and PM yielded reliable kinetic rates across all tested temperatures.

Conclusions:

• Non-equilibrium pulling experiments offer a more robust method for determining DNA hairpin folding parameters than equilibrium hopping experiments, especially in CFM.
• CFM feedback algorithms are more reliable for irreversible pulling experiments, ensuring accurate kinetic rate determination.
• The choice of experimental mode (hopping vs. pulling) and temperature significantly impacts the accuracy of kinetic measurements in optical tweezer studies.