Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article "The Crystal Structure of the GG Reach DNA Quadruplex Sequence GGGGTTTTGGGG in the Presence of Zn2+ and K+ ions" is devoted to the growth and characterization of quadruplex crystals with several different metal ions. This topic is currently relevant for both biochemistry and coordination and crystal chemistry. However, DNA quadruplexes have attracted the attention of researchers since the late 1980s, and the possibility of their stabilization with metal cations has been actively studied since the early 1990s.

There are only a few minor comments about the article:

1. Don't roughly round the values ​​of cell parameters in Table 2 (incorrectly, 47.925 can't be rounded to 47.92) and then provide the corrected values ​​in the text below. Correct the parameter values ​​in the table, and also check the accuracy of the values ​​of other characteristics (in particular, redundancy).
2. In the experimental section (section 2.1) it is stated that "the single-stranded DNA was dissolved in nuclease-free water and annealed by heating at 75 °C for 5 min, followed by slow cooling at room temperature." Explain why heating was done?
3. The meaning of using values [​​1] and [2] in the second column of table S1 is not entirely clear. What does this mean and why is this column not titled?
4. Research on this topic is not limited to studying the crystal structure of the quadruplexes themselves. Therefore, it would be desirable to supplement the introduction with additional references. If necessary, some can be used in the discussion section (at the authors' discretion). A list of suggested sources:

https://doi.org/10.1021/bi982604+

https://doi.org/10.1021/ja709975z

https://doi.org/10.1006/jmbi.1998.2327

https://doi.org/10.1006/jmbi.1996.0039

https://doi.org/10.1007/978-3-319-21756-7_7

https://doi.org/10.1063/1.5050534

https://doi.org/10.1021/ic501484p

Author Response

Response to Reviewer 1

We thank the reviewer for the positive evaluation of our work and for the helpful suggestions. All suggestions have been considered, and the manuscript has been revised accordingly. Detailed responses to each comment are provided below.

Comment 1

Don't roughly round the values ​​of cell parameters in Table 2 (incorrectly, 47.925 can't be rounded to 47.92) and then provide the corrected values ​​in the text below. Correct the parameter values ​​in the table, and also check the accuracy of the values ​​of other characteristics (in particular, redundancy).

Response

Thank you for this remark. The unit cell parameters in Table 2 have been corrected and now match the precision reported in the PDB entry. All crystallographic parameters in the table have been rechecked for consistency with the deposited structure (PDB ID: 9FTA).

Comment 2

In the experimental section (section 2.1), it is stated that "the single-stranded DNA was dissolved in nuclease-free water and annealed by heating at 75 °C for 5 min, followed by slow cooling at room temperature." Explain why heating was done?

Response

The text has been clarified. Heating was used to disrupt any pre-existing secondary structures in the single-stranded DNA and to ensure complete denaturation before refolding. Subsequent slow cooling allows the DNA strands to adopt the thermodynamically favored G-quadruplex conformation.

The following sentence has been added to the manuscript:

Heating was applied to disrupt potential secondary structures and ensure complete strand denaturation before refolding into the G-quadruplex conformation during slow cooling.

Comment 3

The meaning of using values [​​1] and [2] in the second column of Table S1 is not entirely clear. What does this mean, and why is this column not titled?

Response

Thank you for pointing this out. The column heading in Table S1 has now been added. The numbers refer to the type of base pairing:

• [1] – T–T mismatch pairs
• [2] – G–G interactions within tetrads

 

 

Comment 4

Research on this topic is not limited to studying the crystal structure of the quadruplexes themselves. Therefore, it would be desirable to supplement the introduction with additional references. If necessary, some can be used in the discussion section (at the authors' discretion).

Response

Most of the additional references suggested by the reviewer have been incorporated into the Introduction and Discussion sections to support statements regarding cation binding and metal coordination in G-quadruplex structures.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present the information in a somewhat paradoxical way. The structure of the sections, from "Introduction" to "Results", creates a deceptive impression of obtaining an entirely new object. However, in the "Discussion" section, it becomes clear that this discussion merely covers new crystallization conditions for an object whose structure has long been established at high resolution.

Therefore, it seems appropriate to provide this fundamental information in the introduction so that readers are immediately informed.

Minor comments:

Line 95: Specify the starting model.

Lines 109-111: The mention of angle refinement for the P2₁2₁2₁ group is puzzling. By definition, all angles in this orthorhombic group are 90 degrees.

Table 1: Statistical data for the high-resolution shells should be provided.

Figure 2: The 5' and 3' ends should be labeled, and the chains should be colored.

Line 144: Clarification is needed on the designation "AAA-DDD chains." Perhaps A-D was meant?

Comments on the Discussion Section

The presented analysis appears somewhat strange, replete with tables and numerical data. Furthermore, information on coordination error is completely omitted. While this could be considered a minor issue for the 3NZ7 structure (given the 1.1 Å resolution), but for 9FTA at 2.5 Å resolution, and given the extremely poor R-factors (Rwork -33% and Rfree 43%), indicating the extremely low accuracy of the obtained structure, coordination error becomes paramount. In this situation, the abundance of statistical data appears redundant and meaningless. Comparison of the presented structures seems unfounded, especially since the authors compared only one subunit from each of the original structures, whereas in one case there were four, and in the other, two.

 

A much more informative approach would be to present the cross-sectional root mean square deviation (rmsd) for all subunits. This approach clearly demonstrates that the new structure is almost identical to the B subunit in the 3NZ7 structure (standard deviation ≈ 0.4 Å) and significantly different from the A chain in the 3NZ7 structure (standard deviation ≈ 3.6 Å). These are the nuances that should be emphasized. And complemented with a figure with superimposed structures.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer:

Recommendation: This paper may be publishable after major revision; I would like to be invited to further review.

 

Comments:

This manuscript presents a valuable structural characterization of a GG-rich DNA quadruplex in the presence of Zn²⁺ and K⁺ ions, expanding our understanding of G-quadruplex stabilization by mixed metal environments. The data are robust and the crystallographic approach is sound, although some clarity on the biological relevance and generalizability of the findings would strengthen the impact. The discussion would benefit from further emphasis on how Zn²⁺ affects folding and packing compared to monovalent ions. Figures and tables could be improved for clarity, and all referenced supplementary material should be included. The manuscript is generally well written but contains some typographical and formatting inconsistencies that need careful proofreading. Overall, the work is an important contribution to G-quadruplex research, but improvements in data presentation and discussion are recommended. I look forward to the authors’ revisions and clarifications.

Regarding specific questions, I would like the authors to address the following points prior to acceptance:

 

Major comments:

1. How does the biological relevance of this GG-rich sequence and its packing differ from previous G-quadruplex structures? Are there implications for in vivo conditions?
2. Can you expand discussion on how Zn²⁺ and K⁺ ions modulate quadruplex architecture and relate these findings to biological or cellular contexts?
3. Can you include a figure or table with a structural superposition to highlight key differences and similarities with PDB 3NZ7?
4. How do the Zn²⁺ concentrations used compare to physiological conditions? Is the crystallization environment biologically relevant?
5. Can you provide all referenced supporting tables and materials (S1, S2, S3) and summarize the key data for clarity?
6. Is there evidence that mixed ion environments affect quadruplex folding pathways or kinetics, not just static structure?

 

Minor Comments

1. Can you proofread for typographical errors and correct "GG reach DNA" to "GG-rich DNA" in the title?
2. Will you define abbreviations like SCXRD and MPD at first mention?
3. Can you ensure consistent formatting for all references, especially for Items 7, 8, and 9?
4. Can you make the figure legends more descriptive, especially regarding magnification and scale bars?
5. Can you simplify complex sentences for better readability and clarity throughout the manuscript?

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I would move «Molecular replacement was performed using the previously reported G-quad-100 ruplex structure (PDB ID: 3NZ7) as the initial model for structure refinement» to one sentence earlier.

 

Table 1: Statistical data for the high-resolution region should be provided also for rows “reflections “, “I/σ(I)”, “Rmerge % “, “reflections used”, “R (Rfree) %”

”

A resolution range row for refinement statistics should also be added.

Author Response

We thank the reviewer for the careful reading of the manuscript and for the helpful suggestions.

Comment 1:
I would move “Molecular replacement was performed using the previously reported G-quadruplex structure (PDB ID: 3NZ7) as the initial model for structure refinement” to one sentence earlier.

Response:
We thank the reviewer for this suggestion. The sentence describing the molecular replacement procedure has been moved one sentence earlier to improve the text's logical flow.

Comment 2:
Statistical data for the high-resolution region should also be provided for rows “reflections”, “I/σ(I)”, “Rmerge %”, “reflections used”, “R (Rfree) %”.

Response:
We thank the reviewer for this comment. Statistical data for the highest-resolution shell have now been added to Table 2 for the parameters “reflections”, “I/σ(I)”, “Rmerge %”, “reflections used”, and “R (Rfree) %”, in accordance with standard crystallographic reporting practice.

Comment 3:
A resolution range row for refinement statistics should also be added.

Response:
The resolution range used for refinement has now been included in Table 2 to improve the completeness and clarity of the crystallographic data presentation.

Reviewer 3 Report

Comments and Suggestions for Authors

Reviewer:

Recommendation: This manuscript is publishable as it is. 

Comments:

The authors have made significant corrections at appropriate parts of the manuscript. I really appreciate their efforts. I hope it will be published in its current form. 

Author Response

We thank the reviewer for the positive evaluation of our work and for the constructive feedback.

Comment:
The manuscript is publishable as it is. The authors have made significant corrections at appropriate parts of the manuscript. I really appreciate their efforts. I hope it will be published in its current form. 

Response:
We sincerely thank the reviewer for the positive assessment of our manuscript and for recognizing the improvements made during the revision process.