From the journal RSC Chemical Biology Peer review history

25-Jan-2021

Dear Dr Madder, dear Annemieke,

Manuscript ID: CB-REV-12-2020-000236
TITLE: Crosslinker-modified nucleic acid probes for improved target identification and biomarker detection

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,
Andrea (Rentmeister)

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

Reviewer 1

This is a very well-done review on an interesting topic. Furthermore, the topic is not covered in other reviews. Hence, it will be useful to the community. Professor Madder does an excellent job in terms of covering the field. She is also magnanimous in that she does not over emphasize her own group's considerable contributions.
I have only two suggestions:
1. I suggest including Kool's recent "BINARI" probe (https://doi.org/10.1002/anie.202010861), as this is a very original development.
2. The structure are clearly drawn. However, the authors do not consistently represent them with respect to size and proportions. Please make all chemical structures uniform in their presentation.

Reviewer 2

In this review, Joke Elskens and Annemieke Madder described a variety of cross-linkers that can be conjugated to nucleic acids and their applications in the identification and detection of biological targets. Cross-linker modified nucleic acids (capture probes) are especially useful for capturing DNA/RNA targets because they can convert transient probe-target interactions into covalent ones. Therefore, it significantly increases the capture efficiency. This review nicely summarizes the most frequently used cross-linkers, how they can be incorporated into DNA or RNA and their applications in biological systems. It was enjoyable to read this focused and well-written manuscript. I believe it will be of general interest to the chemical biology community as many research laboratories around the world are interested in nucleic acid identification, modification and discovering their interaction partners in different biological contexts.

I suggest that the following points should be clarified before publication:

1. A table summarizing and comparing the key features of the cross-linkers (wavelength, reversibility, cross-linking efficiency, reaction type, target base and position, biological applications, in vivo/in vitro/ex vivo, DNA/RNA etc…) should be added to the review. This table can be very useful for the readers to decide which cross-linker is suitable for their application.

2. At the end of the review, authors can mention challenges and new opportunities for the field. And perhaps talk about the potential features of a dream cross-linker that everybody is looking forward to.

3. Manuscript page 3, Figure 3: this figure should be improved. It is quite hard to understand how the modified NAXCOR method works. There are also some discrepancies between the text and the figure 3B (e.g. the figure shows one cross-link, text says two cross-links). Text says there are cross-links between the green-flanking probes and the red capture probe, which are not shown in the figure. Showing all the details may not be possible in the figure, however this can be clarified in the figure caption (e.g. there are 2 xxx, but only 1 is shown).

4. Manuscript page 4: A generic chemical structure of a psoralen containing nucleic acid should be shown. This can be added into Figure 4 (similar to Fig. 2b for coumarins or Fig. 6 for cyanovinylcarboazoles)

5. Manuscript page 5, “The principle of FISH relies on the use of a molecular beacon that is modified at the 5’ end with a cyanine fluorophore (Cy3) and which contains a dabcyl quencher at the 3’ end (Fig. 7B).” This sentence should be changed. FISH does not rely on the use of molecular beacons; any hybridization probe containing a fluorophore can be used for FISH. However, molecular beacons enable wash-free FISH. Furthermore, this sentence implies that only Cy3 and Dabcyl can be used in molecular beacons. It should, however, read as: “…a molecular beacon that is modified at the 5’ end with a fluorophore (e.g. Cy3) and 3’ end with a quencher (e.g. dabcyl) (Fig. 7B).”

6. Manuscript page 6, left column. It is not clear how thiouridine is used for the identification of protein-RNA interactions (Ref. 69). I assume it is reacting with one of the aromatic amino acids. This should be clarified. Here, the given reference is another review. Original paper should be cited.

7. Manuscript page 7, Figure 10. The chemical structure of the product after step b should be corrected. (-NH is accidentally replaced by -CH2). The legend should be moved to the Figure caption. Step a’ (prime) can be renamed (I find prime confusing) and write down the wavelength number for step a'.

8. Manuscript page 8, Figure 12: A generic chemical structure of a furan containing nucleic acid should be shown.

9. Manuscript page 8, left column: The application of furan-PNAs in combination with photosensitizers can be nicely explained in a figure. It would be so much easier for readers to understand the application.

Minor points:

10. Page 2, right column, “ Additionally, light can travel through space, which in turns enables remote activation.” I think this sentence is not necessary in this context, it is too obvious.

11. Page 2, right column: “This hybridization-based diagnostic test exploits a 7-hydroxycoumarin derivative for photo-crosslinking and is used e.g. for the detection of the factor V…” Remove e.g.

12. Page 4, right column: Spell out RT-qPCR.

13. Human gene names (e.g. p53, BRAF, KRAS) and mRNAs (e.g. FSCN1 and KLF4) should be italic.

14. Page 7, left column, “However, when irradiation with short wavelength UV is required, UV-mediated damage is an often observed and undesirable side effect.” Delete “an”.

15. Page 9, Fig. 14, panel B: The first reaction mechanism arrow should point to the carbonyl group.

Murat Sunbul, PhD
Heidelberg University

This text has been copied from the PDF response to reviewers and does not include any figures, images or special characters.

Dear Editor,
In response to the referee’s comments received at the occasion of our initial submission, we have given these due thought and thus were able to modify and improve the manuscript based on the received feedback. For clarification, the comments we received, and how we have addressed said comments can be found in the 'Response to referees' letter. We strongly believe that our review has been greatly improved as a result of acting on the feedback we received, and would love to have the reviewers agree for final publication.
Thank you for your time, and we look forward to hearing your response.
Please do not hesitate to contact us for further details or additional information, which we will be glad to provide.
Yours Sincerely,
Prof. Dr. A. Madder

REVIEWER REPORT(S):
Referee: 1

Comments to the Author
This is a very well-done review on an interesting topic. Furthermore, the topic is not covered in other reviews. Hence, it will be useful to the community. Professor Madder does an excellent job in terms of covering the field. She is also magnanimous in that she does not over emphasize her own group's considerable contributions.

We thank the reviewer for this positive evaluation of our manuscript and are happy with the compliments given!
I have only two suggestions:
1. I suggest including Kool's recent "BINARI" probe (https://doi.org/10.1002/anie.202010861), as this is a very original development.

Following the reviewer’s suggestion, we included a short discussion on the BINARI probe in the introduction (page 2, left column, yellow highlighted section). As the application lies a bit out of the scope of the review (which focusses specifically on nucleic acid probes modified with a crosslinking agent), we choose to mention it, but did not elaborate too much in detail on the application itself (e.g. we did not include a figure).

2. The structure are clearly drawn. However, the authors do not consistently represent them with respect to size and proportions. Please make all chemical structures uniform in their presentation.
The figures have been adapted to contain chemical structures of uniform size.


Referee: 2

Comments to the Author
In this review, Joke Elskens and Annemieke Madder described a variety of cross-linkers that can be conjugated to nucleic acids and their applications in the identification and detection of biological targets. Cross-linker modified nucleic acids (capture probes) are especially useful for capturing DNA/RNA targets because they can convert transient probe-target interactions into covalent ones. Therefore, it significantly increases the capture efficiency. This review nicely summarizes the most frequently used cross-linkers, how they can be incorporated into DNA or RNA and their applications in biological systems. It was enjoyable to read this focused and well-written manuscript. I believe it will be of general interest to the chemical biology community as many research laboratories around the world are interested in nucleic acid identification, modification and discovering their interaction partners in different biological contexts.

I suggest that the following points should be clarified before publication:

1. A table summarizing and comparing the key features of the cross-linkers (wavelength, reversibility, cross-linking efficiency, reaction type, target base and position, biological applications, in vivo/in vitro/ex vivo, DNA/RNA etc…) should be added to the review. This table can be very useful for the readers to decide which cross-linker is suitable for their application.
Following the reviewer’s fine suggestion, we constructed a table and included it at the bottom of page 2. In this table, crosslinking yields are not reported, as yields are not always mentioned in the original papers describing the applications. Moreover, the crosslinking yield is mostly dependent on the specific biological application and the derivative that is used, so no straightforward and uniform indication can be given concerning the crosslinking yields. We thank the reviewer for the suggestion and are happy with the additional clarity it introduces in our review.

2. At the end of the review, authors can mention challenges and new opportunities for the field. And perhaps talk about the potential features of a dream cross-linker that everybody is looking forward to.
We included an extra paragraph in the conclusion mentioning challenges and new opportunities as well as a tentative description of the ideal crosslinking probe (page 10).

3. Manuscript page 3, Figure 3: this figure should be improved. It is quite hard to understand how the modified NAXCOR method works. There are also some discrepancies between the text and the figure 3B (e.g. the figure shows one cross-link, text says two cross-links). Text says there are cross-links between the green-flanking probes and the red capture probe, which are not shown in the figure. Showing all the details may not be possible in the figure, however this can be clarified in the figure caption (e.g. there are 2 xxx, but only 1 is shown).
We thank the reviewer for pointing this out. The NAXCOR method has now been better illustrated in the figure and discrepancies between the figure and the text have been corrected, clearly improving clarity for the reader.

4. Manuscript page 4: A generic chemical structure of a psoralen containing nucleic acid should be shown. This can be added into Figure 4 (similar to Fig. 2b for coumarins or Fig. 6 for cyanovinylcarboazoles)
In figure 4 (page 4, Fig 4B), examples of psoralen containing nucleic acids have been included.

5. Manuscript page 5, “The principle of FISH relies on the use of a molecular beacon that is modified at the 5’ end with a cyanine fluorophore (Cy3) and which contains a dabcyl quencher at the 3’ end (Fig. 7B).” This sentence should be changed. FISH does not rely on the use of molecular beacons; any hybridization probe containing a fluorophore can be used for FISH. However, molecular beacons enable wash-free FISH. Furthermore, this sentence implies that only Cy3 and Dabcyl can be used in molecular beacons. It should, however, read as: “…a molecular beacon that is modified at the 5’ end with a fluorophore (e.g. Cy3) and 3’ end with a quencher (e.g. dabcyl) (Fig. 7B).”
The text has been changed according to the reviewer’s suggestions.

6. Manuscript page 6, left column. It is not clear how thiouridine is used for the identification of protein-RNA interactions (Ref. 69). I assume it is reacting with one of the aromatic amino acids. This should be clarified. Here, the given reference is another review. Original paper should be cited.
The exact nature of the formed photo-adducts and the mechanism of the photo-reaction is not very-well understood. For this reason, it is not possible to elaborate on the mechanism on the protein-RNA crosslinking reaction. This information was included as a footnote (the extra footnote can be found on page 11). Additionally the original work rather than the review was cited (new ref 70).

7. Manuscript page 7, Figure 10. The chemical structure of the product after step b should be corrected. (-NH is accidentally replaced by -CH2). The legend should be moved to the Figure caption. Step a’ (prime) can be renamed (I find prime confusing) and write down the wavelength number for step a'.
The structure was corrected and the entire figure was adapted according to the reviewer’s suggestions (see new version of figure 10 on page 8.

8. Manuscript page 8, Figure 12: A generic chemical structure of a furan containing nucleic acid should be shown.
Examples of furan containing nucleic acids have been included in the figure (page 9).

9. Manuscript page 8, left column: The application of furan-PNAs in combination with photosensitizers can be nicely explained in a figure. It would be so much easier for readers to understand the application.
A new figure has been included to illustrate the application (figure 12, page 9).

Minor points:

10. Page 2, right column, “ Additionally, light can travel through space, which in turns enables remote activation.” I think this sentence is not necessary in this context, it is too obvious.
This sentence has been removed.

11. Page 2, right column: “This hybridization-based diagnostic test exploits a 7-hydroxycoumarin derivative for photo-crosslinking and is used e.g. for the detection of the factor V…” Remove e.g.
‘e.g.’ is removed

12. Page 4, right column: Spell out RT-qPCR.
RT-qPCR has been spelled out (highlighted in yellow).

13. Human gene names (e.g. p53, BRAF, KRAS) and mRNAs (e.g. FSCN1 and KLF4) should be italic.
Human gene names are italicized (highlighted in yellow).

14. Page 7, left column, “However, when irradiation with short wavelength UV is required, UV-mediated damage is an often observed and undesirable side effect.” Delete “an”.
‘an’ is deleted (sentence highlighted in yellow).

15. Page 9, Fig. 14, panel B: The first reaction mechanism arrow should point to the carbonyl group.
The arrow is adapted and is now pointing to the carbonyl group.

11-Feb-2021

Dear Dr Madder, dear Annemieke

Manuscript ID: CB-REV-12-2020-000236.R1
TITLE: Crosslinker-modified nucleic acid probes for improved target identification and biomarker detection

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, 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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Andrea (Rentmeister)

Reviewer 2

All my questions and suggestions were satisfactorily answered and included in the revised manuscript. The revised manuscript is indeed greatly improved and therefore I recommend the publication of this review without any changes.