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Single-molecule experiments with optical tweezers have become an importanttool to study the properties and mechanisms of biological systems, such ascells and nucleic acids. In particular, force unzipping experiments have beenused to extract the thermodynamics and kinetics of folding and unfoldingreactions. In hopping experiments, a molecule executes transitions between theunfolded and folded states at a preset value of the force (constant force mode-CFM- under force feedback) or trap position (passive mode -PM- withoutfeedback) and the force-dependent kinetic rates extracted from the lifetime ofeach state (CFM) and the rupture force distributions (PM) using the Bell-Evansmodel. However, hopping experiments in the CFM are known to overestimatemolecular distances and folding free energies for fast transitions compared tothe response time of the feedback. In contrast, kinetic rate measurements frompulling experiments have been mostly done in the PM while the CFM is seldomimplemented in pulling protocols. Here, we carry out hopping and pullingexperiments in a short DNA hairpin in the PM and CFM at three differenttemperatures (6$^\circ$C, 25$^\circ$C and 45$^\circ$C) exhibiting largelyvarying kinetic rates. As expected, we find that equilibrium hoppingexperiments in the CFM and PM perform well at 6$^\circ$C (where kinetics isslow) whereas the CFM overestimates molecular parameters at 45$^\circ$C (wherekinetics is fast). In contrast, nonequilibrium pulling experiments perform wellin both modes at all temperatures. This demonstrates that the same kind offeedback algorithm in the CFM leads to more reliable determination of thefolding reaction parameters in irreversible pulling experiments.

Force Feedback Effects on Single Molecule Hopping and Pulling Experiments