Force-Induced Visualization of Nucleic Acid Functions with Single-Nucleotide Resolution
Round 1
Reviewer 1 Report
1. Why was sample well is coated with biotin (first) rather than streptavidin?
2. Provide details of all chemicals used in this study i.e., write manufacturer and Cat. No.
3. Methodology section is hard to understand. To improve the quality of manuscript, authors may include a schematic diagram.
4. Including animation or video of the whole protocol may further improve manuscript quality.
5. How authors validated their approach?
6. Make a comparison between reported approach and previous reports.
Author Response
The authors appreciate the Reviewers’ great effort and scholarly comments regarding this work. We agree with all the comments and have made corresponding modifications to the manuscript. We believe the resulting revision significantly improved the quality and clarity of this manuscript.
The following are our point-by-point responses to the Reviewers’ comments. Their comments are in italic.
Reviewer 1
- Why was sample well coated with biotin (first) rather than streptavidin?
RESPONSE: This is because we have developed a protocol for biotin coating by mixing different percentages of PEG (polyethylene glycol) and biotin-PEG. Optimal ratio has been obtained to maximize the yield of single DNA duplex formation. We have added related information to clarify (Page 4, Line 3 of the “Materials and Methods” section). It is a very valuable idea to directly coat with streptavidin.
- Provide details of all chemicals used in this study, i.e., write manufacturer and Cat. No.
RESPONSE: We have added all the Cat. No. and manufacturer information for purchased chemicals.
- Methodology section is hard to understand. To improve the quality of manuscript, authors may include a schematic diagram.
RESPONSE: We have added a series of schematics to form the flowchart of the whole process in the supporting information (Figure S1). It should be helpful for the readers to understand the method. We appreciate the Reviewer’s nice suggestion.
- Including animation or video of the whole protocol may further improve manuscript quality.
RESPONSE: This is a great idea, which we will certainly consider for all future works. For the current one, the graduate students who performed the majority of the work have long graduated. Nevertheless, we agree with this Reviewer on graphically improving the presentation. So we added a detailed flowchart of the experimental procedure (Figure S1).
- How authors validated their approach?
RESPONSE: Two validations were performed. One is to compare visual detection with optical imaging. The other is to compare with the results from atomic magnetometers. Both the “0” and “1” states are confirmed by the above two different methods (Figure 1). In addition, we expanded the visual method by quantitative absorption measurements (Figure 5).
- Make a comparison between reported approach and previous reports.
RESPONSE: We have added in the discussion section to fairly compare our work with previous work in the field of visual detection (Page 20, Line 6-10).
Reviewer 2 Report
In the present work the authors attempted to develop simple, portable yet cost effective DIY type device to sense nucleic acid sequences. The sensing methodology involves force induced visualization (FIV) based on thermodynamic equilibrium between two competitive hybridizations. The visual/microscope detection of apparent changes of transmitted light intensity has been complemented with atomic magnetometer measurements for force quantification.
The FIV relies on the following factors, (1) magnetically labelled DNA strands (free floating) will bind to the surface immobilized (biotin labelled) complementary analyte strands only, (2) upon binding surface immobilized magnetic labelled particles will produce stronger magnetic signal in comparison to free floating magnetically labelled particles, (3) stronger the magnetic signal, weaker the transmitted light signal would be, (4) successive addition of rulers DNA strands instigates competitive thermodynamic binding/hybridization leading to binary visual detection.
Ruler-to-analyte strand concentration ratio vs mechanical force calibration allows to quantify unknown analyte concentrations. The method has been validated by studying (1) Drug-DNA binding (determining the DNA sequence of interacting sites, interaction strength), (2) mRNA movement during ribosomal translocation.
The thermodynamic stability of DNA hybridization (between reference and analyte strands) determines the strength of magnetic signal. Quantification of the strength of magnetic signal allowed the authors to determine efficacy/relative binding strength of drug-DNA assay. This in turn also useful to intuitively guess relative toxicity of the drugs.
The FIV technique rightfully demonstrates it’s capabilities as an effective analytical technique. Based on this methodology, titrimetric experiments can be designed to determine sequence and concentration of nucleic acids.
Minor comments,
(1) In figure 2a, x-axis captions should be explained properly. Please explain what is ‘R’ & ‘r’. It would be better to write [R]/[A] in the x-axis caption itself. Otherwise, it might be misleading.
(2) Authors claimed to achieve single nucleotide sequence. As a proof negative control experiments should be shown where detection is not successful when two sequences of same length differ by only single nucleotide mismatch.
(3) Mechanical forces need to be determined in order to quantify. However, how exactly mechanical forces are measured is not explained well. Particularly Where exactly atomic magnetometer is placed in the portable device shown in Fig. 5a
(4) The authors did not explain/demonstrate how binding constant of daunomycin is measured.
Author Response
The authors appreciate the Reviewers’ great effort and scholarly comments regarding this work. We agree with all the comments and have made corresponding modifications to the manuscript. We believe the resulting revision significantly improved the quality and clarity of this manuscript.
The following are our point-by-point responses to the Reviewers’ comments. Their comments are in italic.
Reviewer 2
- In figure 2a, x-axis captions should be explained properly. Please explain what is “R” & “r”. it would be better to write [R]/[A] in the x-axis caption itself. Otherwise, it might be misleading.
RESPONSE: Yes, excellent suggestion. We have eliminated R and use directly [R]/[A]. “r” means the relative concentration of the TAM10 buffer, as indicated in the caption.
- Authors claimed to achieve single nucleotide sequence. As a proof negative control experiments should be shown where detection is not successful when two sequences of the same length differ by only single nucleotide mismatch.
RESPONSE: This is a logical consideration. We think the difference between two DNA with consecutive lengths is smaller than the difference between two DNAs with one mismatch. For example, we have previously measured the force difference of an additional A-T pair is only ~8 pN, whereas a single mismatch caused a force difference of 12 pN. Therefore, our contrast between two consecutive DNA rulers is a stronger negative control experiment. But both approaches should work. In that sense, we agree with the Reviewer.
- Mechanical forces need to be determined in order to quantify. However, how exactly mechanical forces are measured is not explained well. Particularly where exactly atomic magnetometer is placed in the portable device shown in Fig. 5a.
RESPONSE: We have added how we calculated the force in the experimental section and provided reference (Page 5, Line 4-6). Atomic magnetometer is not involved in Figure 5a, because it is only used to validate the visual detection method. For Figure 5a, either visual detection or absorption measurements are used.
- The authors did not explain/demonstrate how binding constant of daunomycin is measured.
RESPONSE: We clarified this in the text (Page 16, Line 4 from the bottom).
