From the journal RSC Chemical Biology Peer review history
Dependence of click-SELEX performance on the nature and average number of modified nucleotides
Round 1
Manuscript submitted on 14 Янв. 2022![](https://www.rsc-cdn.org/pubs-core/2022.0.149/content/NewImages/icon-letter.png)
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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
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Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg
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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”.
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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.
Round 2
Revised manuscript submitted on 12 Февр. 2022![](https://www.rsc-cdn.org/pubs-core/2022.0.149/content/NewImages/icon-letter.png)
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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.
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With best wishes,
Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg
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