From the journal RSC Chemical Biology Peer review history

04-Feb-2022

Dear Dr Mayer:

Manuscript ID: CB-COM-01-2022-000012
TITLE: Dependence of click-SELEX performance on the nature and average number of modified nucleotides

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.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to accept your manuscript for publication after revisions. Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

Please submit your revised manuscript as soon as possible using this link :

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a webpage to confirm. ***

https://mc.manuscriptcentral.com/rsccb?link_removed

(This link goes straight to your account, without the need to log in to the system. For your account security you should not share this link with others.)

Alternatively, you can login to your account (https://mc.manuscriptcentral.com/rsccb) where you will need your case-sensitive USER ID and password.

You should submit your revised manuscript as soon as possible; please note you will receive a series of automatic reminders. If your revisions will take a significant length of time, please contact me. If I do not hear from you, I may withdraw your manuscript from consideration and you will have to resubmit. Any resubmission will receive a new submission date.

Supporting our community through Covid-19
While our publishing services are running as usual, we also know that this is a very challenging time for everyone, for many different reasons. If any aspect of the publishing process is worrying you – for example you think you may struggle to meet a pre-determined deadline – please let us know, and we will work out an answer together.

RSC Chemical Biology strongly encourages authors of research articles to include an ‘Author contributions’ section in their manuscript, for publication in the final article. This should appear immediately above the ‘Conflict of interest’ and ‘Acknowledgement’ sections. I strongly recommend you use CRediT (the Contributor Roles Taxonomy from CASRAI, https://casrai.org/credit/) for standardised contribution descriptions. All authors should have agreed to their individual contributions ahead of submission and these should accurately reflect contributions to the work. Please refer to our general author guidelines http://www.rsc.org/journals-books-databases/journal-authors-reviewers/author-responsibilities/ for more information.

The Royal Society of Chemistry requires all submitting authors to provide their ORCID iD when they submit a revised manuscript. This is quick and easy to do as part of the revised manuscript submission process. We will publish this information with the article, and you may choose to have your ORCID record updated automatically with details of the publication.

Please also encourage your co-authors to sign up for their own ORCID account and associate it with their account on our manuscript submission system. For further information see: https://www.rsc.org/journals-books-databases/journal-authors-reviewers/processes-policies/#attribution-id

Please note: to support increased transparency, RSC Chemical Biology offers authors the option of transparent peer review. If authors choose this option, the reviewers’ comments, authors’ response and editor’s decision letter for all versions of the manuscript are published alongside the article. Reviewers remain anonymous unless they choose to sign their report. We will ask you to confirm whether you would like to take up this option at the revision stages.

I look forward to receiving your revised manuscript.

Yours sincerely,
Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg

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

Reviewer 1

This is a very useful paper on the influence of the clicked modifications in DNA libraries (used for click-selex) on the performance of the selex - in particular PCR from the modified DNA. Not suprisingly, the authors found that the more modifications and the more click chemistry, the less efficient the PCR is and the performance of the selex is lower. But the quantification of the effect is very important for practical use of the method. So I recommend this paper for publication with minor revision:
1. in the abstract, the authors should mention the outcome of the study
2. in the conclusions, they should also clearly state the scope and limitations of the technology (which modificantions and how many are safe ...)
3. in the discussion of the PCR from heavily modified DNA templates, they might wich to mention a recent work showing that even fully modified DNA (with all 4 bases modified) can be PCRed and sequenced - see: https://doi.org/10.1093/nar/gkaa999

Reviewer 2

In the manuscript by Siegl et al., the authors systematically investigated the suitability of different chemical moieties for the click-SELEX procedure against C3-GFP as a model target. They also investigated the impact of the clicked-in modifications on the PCR performance by qPCR and compared Ct values obtained from the native DNA library. Finally, the authors analyzed the impact of average number of EdU residues (5.5, 7.0, 9.0) in the library on enrichment during click-SELEX against C3-GFP. Overall, this work exemplifies how a detailed understanding of SELEX procedures can be used to improve results. However, some of the results appear to be rebroadcast of their previous findings in Chem Sci 2020, 11, 9577 and Angew Chem. 2015, 54, 10971, where they have already established that indolyl modifications can improve C3-GFP binding. Extending the present study to some other relevant protein targets would have been more impactful. Additionally, Meyer et al.’s Nature protocols 2018, 13 already provided a detailed procedure and troubleshooting of Click SELEX. Nevertheless, the impact of nucleotide modifications on the various steps of the SELEX process, and in particular PCR, is rarely studied, and thus, this work provides useful insights to the broader aptamer community. The careful analysis of the different SELEX steps by NGS is a highlight of this work. Therefore, I recommend this work for publication after the authors have addressed a few minor comments below:

1. As far as I can tell, the authors do not provide any affinity data on the aptamers identified in this work, instead relying on flow cytometry data to demonstrate binding of individual sequences (Figure 3K). Given that the primary motivation for use of chemical modifications is to improve affinity, it would be useful to know the dissociation constants of these sequences, and in particular, how the different modifications compare. Whether a modification provides a benefit is just as important as its performance during the selection.
2. Figure 3K is listed as G in the caption.
3. Grammatical errors. For example, in page 8 of 33 (or Page 6) “In accordance with this observation is the number of unique sequences…”; gradually to a lesser ‘extend’. Page 10 of 33 (Page 10) “had to stop” was mistyped as “had to stopped”.

Reviewer 1:
in the abstract, the authors should mention the outcome of the study
Answer: We added the following two sentences to the abstract, ‘We demonstrate click-SELEX being strongly depending on which and on how many modifications are used. However, using C3-GFP the number of modifications did not impact the success of the selection procedure.’

in the conclusions, they should also clearly state the scope and limitations of the technology (which modificantions and how many are safe ...)
Answer: We added the following paragraphs to the conclusion section: ‘In most of our previous studies, In-dU, BF-dU, and Bn-dU were used successfully in click-SELEX and found leading to target-specific clickmers.’

in the discussion of the PCR from heavily modified DNA templates, they might wich to mention a recent work showing that even fully modified DNA (with all 4 bases modified) can be PCRed and sequenced - see: https://doi.org/10.1093/nar/gkaa999
Answer: We added the reference in the discussion of the revised manuscript, including the new references 20 and 21. ‘Although it has been shown previously, that fully and modified DNA templates can be amplified by PCR20,21,…’

Reviewer 2:
As far as I can tell, the authors do not provide any affinity data on the aptamers identified in this work, instead relying on flow cytometry data to demonstrate binding of individual sequences (Figure 3K). Given that the primary motivation for use of chemical modifications is to improve affinity, it would be useful to know the dissociation constants of these sequences, and in particular, how the different modifications compare. Whether a modification provides a benefit is just as important as its performance during the selection.
Answer: We thank the reviewer for this important notion. We agree, the impact of chemical modifications on affinity constants of clickmers is an important aspect and in previous studies we could demonstrate that variations in affinity can be found when different click-in moieties have been applied, even post-selectively (refs. 15 & 16: Siegl et al. ACS Chem Biol, 2022; Rosenthal et al. Angew. Chem. 2019). The monoclonal sequences shown in Fig. 3k however, share a specific motif (i.e., CTTTGAATATGTAG) that is related to the previously described clickmer I10 (ref.14: Plückthun et al., Chem. Sci.). We added a sentence to the results section to point this out in the revised version of the manuscript: ‘With exception of clickmer 7EdU(1) these clickmers share a sequence motif (CTTTGAATATGTAG) with the previously identified clickmer I1014, indicating a robust and reproducible enrichment process’

Thus, we did not attempt to do extensive affinity measurements using these very similar clickmers, although variations in affinity may occur but are not expected to be significant. The focus of this study is studying the impact of conditions on the click-SELEX process rather than enriching the best possible click-in target pairs, which has been addressed in ref. 14 from our group.

Figure 3K is listed as G in the caption.
Answer: We thank the reviewer for pointing this out and corrected it in the revised manuscript.

Grammatical errors. For example, in page 8 of 33 (or Page 6) “In accordance with this observation is the number of unique sequences…”; gradually to a lesser ‘extend’. Page 10 of 33 (Page 10) “had to stop” was mistyped as “had to stopped”.
Answer: We corrected the passages accordingly in the revised manuscript.

17-Feb-2022

Dear Dr Mayer:

Manuscript ID: CB-COM-01-2022-000012.R1
TITLE: Dependence of click-SELEX performance on the nature and average number of modified nucleotides

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your revised manuscript for publication. You will shortly receive a separate email from us requesting you to submit a licence to publish for your article, so that we can proceed with the preparation and publication of your manuscript.

You can highlight your article and the work of your group on the back cover of RSC Chemical Biology. If you are interested in this opportunity please contact the editorial office for more information.

Promote your research, accelerate its impact – find out more about our article promotion services here: https://rsc.li/promoteyourresearch.

If you would like us to promote your article on our Twitter account @rsc_chembio please fill out this form: https://form.jotform.com/213543900424044.

Thank you for publishing with RSC Chemical Biology, a journal published by the Royal Society of Chemistry – connecting the world of science to advance chemical knowledge for a better future.

With best wishes,

Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg