Force feedback effects on single molecule hopping and pulling experiments

Single-molecule experiments with optical tweezers have become an important tool to study the prop-erties and mechanisms of biological systems, such as cells and nucleic acids. In particular, forceunzipping experiments have been used to extract the thermodynamics and kinetics of folding andunfolding reactions. In hopping experiments, a molecule executes transitions between the unfoldedand folded states at a preset value of the force [constant force mode (CFM) under force feedback] ortrap position [passive mode (PM) without feedback] and the force-dependent kinetic rates extractedfrom the lifetime of each state (CFM) and the rupture force distributions (PM) using the Bell-Evansmodel. However, hopping experiments in the CFM are known to overestimate molecular distances andfolding 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 CFMis seldom implemented in pulling protocols. Here, we carry out hopping and pulling experiments ina short DNA hairpin in the PM and CFM at three different temperatures (6ºC, 25ºC, and 45ºC)exhibiting largely varying kinetic rates. As expected, we find that equilibrium hopping experimentsin the CFM and PM perform well at 6ºC (where kinetics are slow), whereas the CFM overesti-mates molecular parameters at 45ºC (where kinetics are fast). In contrast, nonequilibrium pullingexperiments perform well in both modes at all temperatures. This demonstrates that the same kind offeedback algorithm in the CFM leads to more reliable determination of the folding reaction parametersin irreversible pulling experiments.