Optical Tweezers to Force Information out of Biological and Synthetic Systems One Molecule at a Time

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

We are very grateful to Rev#1 for the careful reading of the manuscript. We have corrected the indicated typos. We have also expanded the section about the limitations of optical tweezers and described current approaches to overcome these limitations.

Reviewer 2 Report

Bocanegra et al. presents an account of newly developed assays to probe the forces and dynamics of biological systems at the single molecule level. I appreciate that the authors mentioned that they are focusing on assays that were developed in their own lab, which makes this review more focused on the applications that the authors have attempted. I have found the manuscript to be informative and enjoyed some sections of it. However, I found a lack of details as well as textual descriptions of data and experiments that were not part of the figures. I understand that the readers could open the reference papers and see the data, but that is detrimental to the review article. Instead, I encourage the authors to add some details of how the experiments were done and example data for each type of experiment. After all, the abstract says “we present here the assays that we have refined in our laboratory”, but the review in many instances focuses on the results rather than the assays, and relegates the assay description to few sentences without supporting data and schemes. I have pointed out the instances where the authors need, in my opinion, to include more description of the assays and more data in the figures to paint a clear picture of what these experiments are. This will do justice to the nice experimental approaches that the authors are describing. Overall, I think the substance of this review is worthy of publication and the article deserves to be published once these few comments are addressed. I list my comments below along with some minor grammar and spelling errors that I found in the manuscript.

 

Major comments:

- Page 3 line 128: the authors state that “In collaboration with the lab of Prof. Fernando Moreno-Herrero (CNB-CSIC), we found that these sequences are remarkably soft at low forces (P~20nm) and stiff at high forces (S~2400nm).”. I find no paper that is referenced nor is the data presented in the figure. I suggested the authors elaborate on what they mean by low and high forces. Technically the DNA is stretched at a constant velocity and the force applied by the DNA tether on the bead is what is reported in an OT experiment. So the difference between the 20 nm forces and the 2400 nm forces experiments is not clear, is it different stretching velocities that the authors have used or different end-to-end distance? or is it a different trap stiffness ?. Further, the authors need to present the data for the low and high forces and show how the stiffness of polyA-rich DNA tracts changes. The authors further need to report what is the type of DNA used here?

  

- Line 273-277: The authors need to explain how they came to the conclusions of the two major binding modes with 35 nt and 65 nt wrapping of DNA. This review was introduced as an example of how OTs extract information out of biological systems. But the description of the sections focuses too much on the results and not on the method (the How? Part). It would be more beneficial to the reader if the authors briefly describe how can you quantify the number of nucleotides that are wrapped via binding of a certain protein (N_nt) with some example data (force curves) that elucidate the methodology.

 

- Lines 322-328: The authors need to put a figure here. At minimum: a cartoon of the DNA structure as tethered by the two optical traps. An example data of how the activity was measured (end-to-end distance variation with time) in the presence and absence of mtSSB, and some data to elucidate how the kinetics are limited by the reannealing of the DNA and the applied force.
Are all these findings in reference 66? If so, then the reference should be cited again within the paragraph.

 

- Line 366: can the authors show an example of a force curve where fission occurs?

- Lines 387 and onwards: The authors need to show the data they are talking about, otherwise it is very difficult for the reader to understand the results they are portraying about how Dyn2 eliminates the retraction force of the membrane. A figure that is similar to figure 5 would be really helpful. Otherwise, I fear that readers will glance over these results since no data is being shown.

 

- Kudos for the authors on writing section 6 of the article. It is well-written with enough details and figures that makes it easy to read and understand. I encourage the authors to go through the previous sections and add the details that I mentioned in my previous comments to bring the level of the previous sections to that of section 6, which was a joy to read!

 

- The authors might want to include a paragraph on the limitations of optical tweezers and their applications in cells. The difficulty (or not) of tweezer calibration and whether there are examples of applying OT in cells to perform single molecule experiments. This is a mild suggestion.

 

 

 

Minor comments:

 

Line 130: Grammar error. “this imply” should be “this implies”.

Line 131: “Thi finding” should be “this finding”

Line 178: “Single-molecule manipulations, as OT and magnetic tweezers have provided” should be “Single-molecule manipulations, as OT and magnetic tweezers, have provided”

Line 183: “motionve” spelling error.

Lines 266 and 267 need a reference.

Line 313 and 329: “polg” is a spelling error.

Line 339: “besides to study DNA-based motors” should be “besides studying DNA-based motors”.

Line 355: what does NT stand for?

Line 390: “fiission” spelling error

Line 496: “placed then” should be “placed them”

Figure 3B: the sign of the forces in the legend is not defined. Which ones are the aiding forces, positive or negative?

 

Author Response

We are very grateful to Rev#2 for the careful reading of the manuscript. We detailed below a point-by-point response.

Major comments:

- Page 3 line 128: the authors state that “In collaboration with the lab of Prof. Fernando Moreno-Herrero (CNB-CSIC), we found that these sequences are remarkably soft at low forces (P~20nm) and stiff at high forces (S~2400nm)”. I find no paper that is referenced nor is the data presented in the figure. I suggested the authors elaborate on what they mean by low and high forces. Technically the DNA is stretched at a constant velocity and the force applied by the DNA tether on the bead is what is reported in an OT experiment. So the difference between the 20 nm forces and the 2400 nm forces experiments is not clear, is it different stretching velocities that the authors have used or different end-to-end distance? or is it a different trap stiffness ?. Further, the authors need to present the data for the low and high forces and show how the stiffness of polyA-rich DNA tracts changes. The authors further need to report what is the type of DNA used here?

We thank Rev#2 for pointing out these issues. We agree that an additional figure describing the experiments with the A- tracts may help the reader to understand this section. However, we note that although we collaborated in the work to study the mechanical properties of A-tracts, the original ideas and the experimental designs for these studies did not come from our lab. Therefore, we considered that their inclusion in a review that describes optical tweezers experimental designs developed in our lab was not fair. In an attempt to solve this issue, we have summarized the section regarding the A-tracts. In the revised version we now indicate only the magnitude of the parameters that define the mechanical properties of these specific dsDNA sequences (page 3, last paragraph).

We note that we included the section ‘Optical tweezers to interrogate the elastic properties of DNA’ to inform the reader about the relevance to determine the mechanical properties of ds- and ss- DNA for the proper analysis of the results described in the following sections.

- Line 273-277: The authors need to explain how they came to the conclusions of the two major binding modes with 35 nt and 65 nt wrapping of DNA. This review was introduced as an example of how OTs extract information out of biological systems. But the description of the sections focuses too much on the results and not on the method (the How? Part). It would be more beneficial to the reader if the authors briefly describe how can you quantify the number of nucleotides that are wrapped via binding of a certain protein (N_nt) with some example data (force curves) that elucidate the methodology.

Following Rev#2 suggestion, in the revised version of the manuscript we explain that the number of nucleotides bound per SSB tetramer were quantified by fitting the force-extension curves of the SSB-DNA polymers to a theoretical model that explains the mechanics of ligand binding to biopolymers (Jarillo et al, new reference 65). We also have included a new Figure (Figure 4B) showing a representative force-extension curve of the SSB-DNA polymers fitted to the theoretical model.

- Lines 322-328: The authors need to put a figure here. At minimum: a cartoon of the DNA structure as tethered by the two optical traps. An example data of how the activity was measured (end-to-end distance variation with time) in the presence and absence of mtSSB, and some data to elucidate how the kinetics are limited by the reannealing of the DNA and the applied force.
Are all these findings in reference 66? If so, then the reference should be cited again within the paragraph.

We agree with Rev#2 that a figure would be needed in this section. We did not include figures to explain these experiments and/or show these results because the work described in this paragraph is not yet published, it is currently under review at NAR. To solve this issue, in the revised version of the manuscript, we indicate that we are currently studying the DNA unwinding activities of the mitochondrial DNA polymerase and helicase using an experimental set up similar to that descried in Figure 3D. We finally decided not to include these unpublished data, which cannot be properly cited.

- Line 366: can the authors show an example of a force curve where fission occurs?

According to Rev#2 suggestion we have included an experimental curve showing fission; Figure 5C in the revised manuscript. .

- Lines 387 and onwards: The authors need to show the data they are talking about, otherwise it is very difficult for the reader to understand the results they are portraying about how Dyn2 eliminates the retraction force of the membrane. A figure that is similar to figure 5 would be really helpful. Otherwise, I fear that readers will glance over these results since no data is being shown.

We agree with Rev#2 that a figure showing our data on the membrane remodeling activity of Dyn2 would indeed be very helpful to understand the results. However, this data is still not published. As we did before, we decided to exclude this data from the review. In the revised version we mention only that an optical tweezers experimental setup similar to that described to study the effect reticulon protein on membrane nanotubes (Figure 5) offers the possibility to study membrane remodeling dynamics by Dyn1 and Dyn2 proteins.

We are very thankful to Rev#2 for calling our attention to this issue.

- Kudos for the authors on writing section 6 of the article. It is well-written with enough details and figures that makes it easy to read and understand. I encourage the authors to go through the previous sections and add the details that I mentioned in my previous comments to bring the level of the previous sections to that of section 6, which was a joy to read!

Thank you.

- The authors might want to include a paragraph on the limitations of optical tweezers and their applications in cells. The difficulty (or not) of tweezer calibration and whether there are examples of applying OT in cells to perform single molecule experiments. This is a mild suggestion.

According to Ref#2 suggestion, we have expanded our description of the limitations of optical tweezers and included a short paragraph about their application to study molecular reactions in cells.

Round 2

Reviewer 2 Report

I thank the authors for addressing my points adequately. I have no further comments on the revised manuscript and recommend it for publication.