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Force feedback effects on single molecule hopping and pulling experiments.

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Optical tweezers hopping experiments in constant force mode (CFM) can overestimate DNA hairpin folding parameters at high temperatures. Non-equilibrium pulling experiments in CFM and passive mode (PM) provide reliable kinetic rates across temperatures.

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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.