From the journal RSC Chemical Biology Peer review history

03-Mar-2024

Dear Dr Di Antonio:

Manuscript ID: CB-REV-01-2024-000023
TITLE: Genome-wide mapping of G-quadruplex DNA: a step-by-step guide to select the most effective method.

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Dr Andrea Rentmeister

************

Reviewer 1

G-quadruplexes (G4) are unusual DNA structures that dynamically form in guanosine-rich regions throughout the human genome. The protruding loop sequence determines the structural diversity and thermodynamic stability of G4 and is conceivably affected by the respective G4's local chromatin environment. Bioinformatic studies find G4s are abundant in regions with tumor-biology significance, such as oncogene promoter regions. The development of the first chromatin-compatible G4 antibody by the Balasubramanian group (BG4) opened the era of G4 functional genomic studies. BG4 immunoprecipitation of G4-chromatin coupled with next-generation sequencing (ChIP-seq) successfully identified shared and unique genomic loci between human non-transformed and cancer cells and confirmed specific enrichment of oncogene promotor regions in cancer cell samples. Following this success, increasingly higher resolution and orthogonal approaches are described to enrich the data and provide the current landscape of G4's importance in different biological processes.

In this comprehensive review, the authors systematically evaluate the existing methodologies for genome-wide mapping of G4. The authors did a great job setting the stage by providing a historical context of the methodologies' development and discussing in detail the limitations of these methods in translating to meaningful biological understandings. The review is well-written, with sufficient details and insightful conclusions to capture a broad audience within and beyond the immediate G4 biology fields. This reviewer congratulates the authors on a good job and recommends publication of this work, with one caveat: the authors specifically stated that their primary aim is to "<i>offer a step-by-step guide to the readers in selecting the most appropriate strategy that fits best their experimental needs.</i>" This reviewer believes that including a diagram that provides a decision tree to quickly summarize the authors' recommendation based on downstream data usage and applications would be much more appreciated by the non-expert audience than the current Figure 7, which only recapitulates the assays' features. There are also a few minor typos, formatting errors, and a couple of suggestions that the authors may consider incorporating into the final manuscript to enrich the "what is next" discussions. However, given that these are aspirational (no data yet), the authors should consider these suggestions optional. These are listed in point form below:

Typos and Formatting errors:
1) In Figure 2, the heading for G4-ChIP-Seq is misspelled as G4-ChIP-Sep

2) The manuscript has repeating gaps and formatting errors, which should be easily corrected at the copy-editing stage. However, I want to list some of them here briefly:
• page 4, the last sentence of the page has a huge gap
• page 5, an entire column of text is missing on the right
• page 10, the formatting divides the page horizontally and then vertically, which is not common

Suggestions:
1) while the reviewer appreciates the development of a smaller and more versatile antibody by the senior author's group (SG4), there is redundancy in the description and lauding of the benefits of this antibody throughout the manuscript. The authors may consider streamlining these discussions for a more concise presentation.

2) the authors include experiments using biotinylated PhenDC3 and PDS in the mapping discussions using chemical probes. The family of TASQ molecules should also be included in this discussion for completion.

3) this reviewer agrees with the authors that G4 access remained lacking as a direct G4-discovery approach. The primary paper showed that sub-nucleosome fragments protected from MNase digestion are not all necessarily G4s, ascertaining the need for complementary methods to prove that the G4access peaks are indeed G4s. Could the authors highlight this as a significant concern, specifically in the summary figure (figure 2)? Without this highlight, G4access reads favourably as a method that is not dependent on probe/antibodies and thus is free from the binding affinity bias associated with these methods.

4) This reviewer also agrees with the authors on the need to develop new capturing methods to facilitate the studies of long-range and inter-molecular G4. To enrich this discussion, would the authors consider adding how current genomic methods such as hi-c-seq could be adapted for the study of G4s?

Reviewer 2

This is an exceptional review that covers whole genome mapping of G-quadruplex DNA structures. This is a highly topical area in the field of G-quadruplexes and the authors have provided a very well-justified and thought out review of the whole field to date. Although there may indeed be more techniques along the way, this is a good time for the review to be published and I am supportive of publication in its current form following a superficial revision for English language.

The title is informative and explicit about the content and will enable readers to easily identify the purpose of the review. The figures are well thought out, and appealing to the eye. The narrative throughout the review is good and easy to follow for a new reader. Figure seven is a good overview for the reader and helps users select which method is most appropriate for their situation.

There are a number of typographical errors, but these are very minor and I expect these will be picked up in RSC's proofing process, or if other changes are requested from reviewers then the authors should consider using a grammar checker or Chat GPT to polish the English language and correct the spacing. Exemplar required edits, as an example from the abstract are:
"we sought to" should be either "we have sought to" or "we seek to"
strenghts should be strengths
"and select the most" should be "and selecting the most" or "and how to select the most"

I would recommend all abbreviations are defined at first use. E.g. ChIP as part of ChIP-seq

Overall this is a very nice review and wholly suitable for RSC Chemical Biology

REVIEWERS RESPONSE

Referee: 1

G-quadruplexes (G4) are unusual DNA structures that dynamically form in guanosine-rich regions throughout the human genome. The protruding loop sequence determines the structural diversity and thermodynamic stability of G4 and is conceivably affected by the respective G4's local chromatin environment. Bioinformatic studies find G4s are abundant in regions with tumor-biology significance, such as oncogene promoter regions. The development of the first chromatin-compatible G4 antibody by the Balasubramanian group (BG4) opened the era of G4 functional genomic studies. BG4 immunoprecipitation of G4-chromatin coupled with next-generation sequencing (ChIP-seq) successfully identified shared and unique genomic loci between human non-transformed and cancer cells and confirmed specific enrichment of oncogene promotor regions in cancer cell samples. Following this success, increasingly higher resolution and orthogonal approaches are described to enrich the data and provide the current landscape of G4's importance in different biological processes.

1) In this comprehensive review, the authors systematically evaluate the existing methodologies for genome-wide mapping of G4. The authors did a great job setting the stage by providing a historical context of the methodologies' development and discussing in detail the limitations of these methods in translating to meaningful biological understandings. The review is well-written, with sufficient details and insightful conclusions to capture a broad audience within and beyond the immediate G4 biology fields. This reviewer congratulates the authors on a good job and recommends publication of this work, with one caveat: the authors specifically stated that their primary aim is to "<i>offer a step-by-step guide to the readers in selecting the most appropriate strategy that fits best their experimental needs.</i>" This reviewer believes that including a diagram that provides a decision tree to quickly summarize the authors' recommendation based on downstream data usage and applications would be much more appreciated by the non-expert audience than the current Figure 7, which only recapitulates the assays' features. There are also a few minor typos, formatting errors, and a couple of suggestions that the authors may consider incorporating into the final manuscript to enrich the "what is next" discussions. However, given that these are aspirational (no data yet), the authors should consider these suggestions optional. These are listed in point form below:

We changed the Figure 7 to render it easier for non-expert researchers to choose the best method to approach G4 mapping, as suggested by the reviewer. In particular we replaced the voice "Fixation" with a more informative "Sample availability" (High vs Low). However, we believe that a decision tree structure would limit the breadth of this figure. Each method presented in this review has advantages and disadvantages depending on the context, thus a decision tree would be very limiting. By looking at a figure that is closer to a summary of the properties for each technique, the reader can select the best approach in a more informative way according to their specific needs, as also highlighted by reviewer 2.

2) In Figure 2, the heading for G4-ChIP-Seq is misspelled as G4-ChIP-Sep

We have corrected this typo as suggested.

3) The manuscript has repeating gaps and formatting errors, which should be easily corrected at the copy-editing stage. However, I want to list some of them here briefly:
• page 4, the last sentence of the page has a huge gap
• page 5, an entire column of text is missing on the right
• page 10, the formatting divides the page horizontally and then vertically, which is not common

We apologise for the formatting errors that have been embedded during the uploading of the original word document. We have now fixed those accordingly.

4) while the reviewer appreciates the development of a smaller and more versatile antibody by the senior author's group (SG4), there is redundancy in the description and lauding of the benefits of this antibody throughout the manuscript. The authors may consider streamlining these discussions for a more concise presentation.

We agree with the reviewer that some of the SG4 discussion was repeated thorough the manuscript. We have now removed redundant comments on the G4-probe throughout the manuscript.

5) The authors include experiments using biotinylated PhenDC3 and PDS in the mapping discussions using chemical probes. The family of TASQ molecules should also be included in this discussion for completion.

We have now included TASQ molecules as possible tools to be used in Chem-map.

6) this reviewer agrees with the authors that G4 access remained lacking as a direct G4-discovery approach. The primary paper showed that sub-nucleosome fragments protected from MNase digestion are not all necessarily G4s, ascertaining the need for complementary methods to prove that the G4access peaks are indeed G4s. Could the authors highlight this as a significant concern, specifically in the summary figure (figure 2)? Without this highlight, G4access reads favourably as a method that is not dependent on probe/antibodies and thus is free from the binding affinity bias associated with these methods.

As we already discuss in the main text, we also strongly agree on the limitations around G4access. We have now modified the G4access voice in Figure 2 by adding a bullet point in which we highlight that G-rich regions identified by G4access require further in vitro validation.

7) This reviewer also agrees with the authors on the need to develop new capturing methods to facilitate the studies of long-range and inter-molecular G4. To enrich this discussion, would the authors consider adding how current genomic methods such as hi-c-seq could be adapted for the study of G4s?

A short comment on the potential use of Hi-C to map G4s in the 3D genome has been added as requested.

Referee: 2

This is an exceptional review that covers whole genome mapping of G-quadruplex DNA structures. This is a highly topical area in the field of G-quadruplexes and the authors have provided a very well-justified and thought out review of the whole field to date. Although there may indeed be more techniques along the way, this is a good time for the review to be published and I am supportive of publication in its current form following a superficial revision for English language.
There are a number of typographical errors, but these are very minor and I expect these will be picked up in RSC's proofing process, or if other changes are requested from reviewers then the authors should consider using a grammar checker or Chat GPT to polish the English language and correct the spacing. Exemplar required edits, as an example from the abstract are: "we sought to" should be either "we have sought to" or "we seek to" strenghts should be strengths "and select the most" should be "and selecting the most" or "and how to select the most"
I would recommend all abbreviations are defined at first use. E.g. ChIP as part of ChIP-seq

We thank the Reviewer for their comments. Typographical, grammatical, and formatting errors have bene amended as suggested.

21-Mar-2024

Dear Dr Di Antonio:

Manuscript ID: CB-REV-01-2024-000023.R1
TITLE: Genome-wide mapping of G-quadruplex DNA: a step-by-step guide to select the most effective method.

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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Reviewer 1

The authors did a great job revising the manuscript and successfully addressed all my comments, their efforts are much appreciated. I have no further concerns.