Detecting Molecular Folding from Noise Measurements

Round 1

Reviewer 1 Report

In this work, Rico-Pasto and Ritort et al studied the detection of molecular folding from noise. This is an elegant and clean work which extend the application of Brownian theory in the single-molecule area. The theory is supported by the DNA hairpin measurements using OT technique. Therefore, the reviewer strongly recommends this work to be published after minor revisions, and is not necessary to send it back to the reviewer for the 2nd round.

In a previous single-molecule study on the anisotropic friction in a ligand-protein complex by Wolf and Balzer et al, the authors prove that the friction coefficient is positively related to the force variance, and the force fluctuation is highest around the position of energy barrier. It would be good to refer to this work, if this is related to the authors' findings.

In a typical single molecule pulling experiment, the noise level is strongly related to the calibration of the stiffness of the pulling device, and usually accompained with an random error. How is the stiffness calibrated, and would this bring any effect in this work?

Please comment the effects of pulling velocity (loading rate) in this situation.

It is better to briefly compare the unfolding force in this study with the previous unfolding force of hairpins as a reference for the validation.

Author Response

We thank the reviewer for her/his positive recommendation for publication. In what follows, we will answer the points raised by the reviewer.

Concerning the first question raised by the reviewer, the work done by Wolf and Balzer et al., demonstrated that the rupture force variance of the ligand-protein complex biotin-streptavidin increases close to the transition state. In contrast, our work is focused on how the force variance remains at a given stable state. All this said, we found these results interesting and added a new paragraph in the introduction to highlight these findings.

“Recently, using single-molecule experiments it has been demonstrated that the rupture force variance of the ligand-protein complex biotin-streptavidin increases close to the transition state [8].”

Regarding the calibration of the instrument stiffness, different methods are employed depending on how the instrument operates. In our case, the force signal is measured directly from the light deflection after passing through the trapped bead. It means that the force is measured without assuming any model, e.g., Hooke’s law. We calibrate the force signal using a Stokes test. A bead of known dimensions is trapped with the optical tweezers and moved along one direction at different velocities in a microfluidic chamber filled with pure water. Under these conditions, the force is proportional to the moving speed. Therefore, to know the trap stiffness (), we commonly conduct one of these two experiments: 1) study the power spectrum of the force signal when a bead is located in the optical trap at zero force. 2) displace the trapped bead a given distance relative to the center of the optical trap and measure the force. This means that we can measure not only the trap stiffness but also, how it changes at different forces. Then, how does the trap stiffness change upon pulling experiments? In a previous work, the laboratory investigated this question (new reference 25 in the manuscript), finding that the trap stiffness changes less than 1% on the explored force range. For this reason, in the manuscript, we take  as constant.

Finally, regarding the suggestion made by the reviewer to compare the experimental unfolding forces for the explored DNA hairpins with existing literature, we want to remark that the DNA sequences used in this work have been studied in the explored range of temperatures for the first time in the present manuscript. Therefore, we do not have an experimental work, using a temperature-dependent optical tweezer or other single-molecule instrument, to compare with our data.

Reviewer 2 Report

The manuscript entitled “Detecting molecular folding from noise measurements” by Rico-Pasto et al has demonstrated the detection of folding events at low forces. The results are very interesting. Following minor changes are required before publication:

1.       The sentence in line 3, page 3 is misleading. Authors should rephrase the sentence to clarify the change of forces during the pulling experiment.

2.       Authors should change the figure caption, specifically the way “Dark (light)…….unfolding(folding)” are mentioned.

Author Response

We thank the reviewer for her/his positive recommendation for publication and the comments that helped to improve the manuscript.

The first sentence that the reviewer recommended change now reads, “In a pulling cycle, the force applied to the system increases (decreases) when moving the optical trap away (towards) to the pipette.”

Finally, the caption of Figure 1 now reads, “The dark color lines denote the unfolding trajectories, while the light color lines correspond to folding trajectories.”