  Single-molecule experiments with optical tweezers have become an important
tool to study the properties and mechanisms of biological systems, such as
cells and nucleic acids. In particular, force unzipping experiments have been
used to extract the thermodynamics and kinetics of folding and unfolding
reactions. In hopping experiments, a molecule executes transitions between the
unfolded and folded states at a preset value of the force (constant force mode
-CFM- under force feedback) or trap position (passive mode -PM- without
feedback) and the force-dependent kinetic rates extracted from the lifetime of
each state (CFM) and the rupture force distributions (PM) using the Bell-Evans
model. However, hopping experiments in the CFM are known to overestimate
molecular distances and folding free energies for fast transitions compared to
the response time of the feedback. In contrast, kinetic rate measurements from
pulling experiments have been mostly done in the PM while the CFM is seldom
implemented in pulling protocols. Here, we carry out hopping and pulling
experiments in a short DNA hairpin in the PM and CFM at three different
temperatures (6$^\circ$C, 25$^\circ$C and 45$^\circ$C) exhibiting largely
varying kinetic rates. As expected, we find that equilibrium hopping
experiments in the CFM and PM perform well at 6$^\circ$C (where kinetics is
slow) whereas the CFM overestimates molecular parameters at 45$^\circ$C (where
kinetics is fast). In contrast, nonequilibrium pulling experiments perform well
in both modes at all temperatures. This demonstrates that the same kind of
feedback algorithm in the CFM leads to more reliable determination of the
folding reaction parameters in irreversible pulling experiments.
