From the journal RSC Chemical Biology Peer review history

03-Jan-2024

Dear Dr Abe:

Manuscript ID: CB-ART-10-2023-000212
TITLE: Development of PCR Primer Enabling the Design of Flexible Sticky Ends for Efficient Concatenation of Long DNA Fragments

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Associate Editor, RSC Chemical Biology

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

Reviewer 1

The work introduces a new entry in the stop primer method for creating sticky ends on DNA. It uses chemically modified primers to stop the extension during the PCR, allowing the formation of sticky ends that can be used for high efficiency DNA ligation. While there are other approaches to the technique in the literature, this one might prove a valuable new addition if the phosphoramidites can be readily available for researchers.

Here are some points that should be addressed before publication:
1. little mistake in figure 1 : change "Adamamtyl" to "Adamantyl"
2. for the plasmid construction, it would be nice to perform a Sanger sequencing experiment to check the potential mutations on ligated products. I see that the product is only checked by gel to see the correct size.
3. A comparison with the existing cleavable group shown in the figure 1 and the o-nitrobenzyl group could be added.

Reviewer 2

The manuscript details the design and application of PCR stop primers with phosphate-caged nucleotides, presenting a collection of fifty-four uniquely designed and synthesized primers. The same concept was originally reported by Kuzuya and Komiyama, as noted by the authors in the text (refs. 36-39). The present report successfully overcomes the limitation of the previous reports by introducing the photocages into the phosphate rather than the nucleobase. The synthetic methods and chemical properties are meticulously planned and articulated. The innovative concept of photolabile stop primers is demonstrated for constructing sticky ends of precise lengths. The results are well organized and provide enough evidence to attract interest in chemical biology from peers. Considering that the manuscript was transferred to this journal based on the reviewers' comments in Chemical Science, I would like to support the publication of this manuscript in this journal. While I find the manuscript suited for publication, I propose some adjustments for your consideration:

(1) In Figure 3, the native PAGE images exhibit stains that seem to replicate across different experiments, particularly noted as diagonal lines running from the upper left to the lower center in images b, g left, and h. Could you clarify the origin of these identical stains? If these are artifacts and the original fluorescent images are clear, I would recommend substituting the current images with the unblemished versions.

(2) Regarding Figure 3, in some lanes, the same locations as the PCR products in template 23 appear distinctly whiter than the background. Quantifying the fluorescence intensity in this region would be essential to assess the stopping efficiency accurately. Could you provide an explanation or further clarification on this observation?

(3) In Table 1, the listed sequence for ODN_25 appears to contain an additional 'C' compared to its description in Figure 3. Would you mind verifying this sequence and updating the table accordingly to ensure accuracy?

(4) While you have compared the DNA linkage efficiency to that of the restriction enzyme method, it might be beneficial to also compare it to other methods like In-Fusion cloning, which improves ligation efficiency through the addition of extended adhesive ends via PCR. Including such a comparison could significantly underscore the unique advantages of your method.

I appreciate the opportunity to review this study and look forward to your revisions that further refine this promising research. Thank you for considering my suggestions.

Reviewer 3

A very interesting work about photocaged primers for DNA concatenation is reported in the manuscript. Although the photocaging chemistry is not completely new, it is clearly demonstrated that the concatenation method reported by the authors is advantageous in directly converting long DNA fragments into even longer products. I support publication of this manuscript in RSC Chem Biol after a minor revision addressing the following comments:

(1) The decaging product analyses in Figure 2 are based on only one technique, HPLC retention time. These data alone can only prove that there are some chemical changes occurred after light irradiation. I recommend ESI-MS analyses on the decaging products (those in Figure 2b and 2c) to confirm they are indeed the desired products, excluding the possibilty of incomplete decaging or any photo-induced damage to the DNA products. In addition, I recommend the authors to provide their ESI-MS data chart (images), instead of only numbers in tables.

(2) Because 365 nm UV light is applied in the method, there is possibility of photo-induced damages to the DNA, and the consequence may be more servere (such as wrong protein translation) when the DNA fragments are long. I recommend the authors using either the plasmid (Figure 5a) or the long DNA product (Figure 5b) to do a Sanger sequencing analysis, to show that the product sequence is indeed very clean.

Referee: 1
Thank you very much for your time and efforts in reviewing our manuscript. We sincerely appreciate your favorable evaluation of our method for synthesizing DNA fragments with sticky ends. Your comments were very constructive and allowed us to improve the quality of our manuscript. We have incorporated additional experimental data and necessary revisions. The changed or added parts are highlighted in yellow. We hope that these revisions meet your expectations and that the revised manuscript is now satisfactory. Your feedback has been invaluable to us, and we are grateful for your contribution to enhancing our work.

Comment_1
Here are some points that should be addressed before publication:
1. little mistake in figure 1 : change "Adamamtyl" to "Adamantyl"

Response_1
Thank you for pointing out the spelling error in 'Adamantyl' in Figure 1. This was indeed our oversight. We have replaced Figure 1 with the corrected spelling of 'Adamantyl.'

Comment_2
2. for the plasmid construction, it would be nice to perform a Sanger sequencing experiment to check the potential mutations on ligated products. I see that the product is only checked by gel to see the correct size.

Response_2
Thank you for your feedback regarding the sequence determination experiments during plasmid construction. We appreciate your suggestion. In response, we selected 16 blue colonies obtained in Figure 5a, conducted culturing and plasmid extraction, and performed sequence analysis using the Sanger method. As a result, among the 32 junction sites, we observed mutations at 3 locations. These observed mutations included instances of mismatch mutations during gap-filling by the polymerase during ligation, as well as cases where bases were skipped. In contrast, no mutations were observed in regions other than junction sites. Nevertheless, in most cases, we confirmed the successful construction of the plasmids with the correct sequences.

We have incorporated this information into the main text as follows: [Sixteen blue colonies were picked and cultured, and plasmids were extracted from them. The sequences of the obtained plasmids were analysed using the Sanger method. The results revealed that mutations occurred at three of the 32 ligation sites (Fig. S3). The observed mutations were mismatch mutations that occurred during gap-filling by the polymerase, or base skipping. However, the construction of plasmids with the correct sequence was confirmed in most of the samples. In addition, no mutations were observed in regions other than the ligation site. Thus, the 365 nm photoirradiation used to remove the o-nitrobenzyl modification did not induce DNA mutations.]. Furthermore, we have included a new section in the Supporting Information titled 'Sequence analysis of constructed plasmid' to provide a detailed account of the sequence analysis procedure. The results of the sequence analysis have been added as Figure S3. Thank you for your valuable input.

Comment_3
3. A comparison with the existing cleavable group shown in the figure 1 and the o-nitrobenzyl group could be added.

Response_3
Thank you for your important feedback. We acknowledge that we cannot provide direct data comparing our o-nitrobenzyl protecting group with other protecting groups mentioned in Figure 1, as those other protecting groups are not available to us. Therefore, we intend to discuss and compare our results with existing literature information.

In the case of the existing 4-O-[2-(2-nitrophenyl)-propyl]thymine protecting group, it has been reported to have a protection cleavage efficiency of approximately 70% upon light irradiation.36 On the other hand, our protecting group, as revealed in this study, is shown to be removed with nearly 100% efficiency. Therefore, in terms of deprotection efficiency, it is believed that our method surpasses conventional approaches. We have included this information in the main text to emphasize the superior deprotection efficiency of our protecting group as compared to the previously reported one. We have incorporated this information into the main text as follows: [The above results demonstrate that this chemical modification exhibits a higher deprotection efficiency compared to the existing 4-O-[2-(2-nitrophenyl)-propyl]thymine.36].

Referee: 2
Thank you very much for your valuable time and effort in reviewing our manuscript. We sincerely appreciate your favorable evaluation of our method for synthesizing DNA fragments with sticky ends. Your comments were instrumental in improving the quality of the manuscript, as evidenced by the revisions and data replacements outlined in Responses_1-3. The changed or added parts are highlighted in yellow. We hope that these revisions meet your expectations and that the revised manuscript is now satisfactory. Your feedback has been invaluable to us, and we are grateful for your contribution to the enhancement of our work.

Comment_1
(1) In Figure 3, the native PAGE images exhibit stains that seem to replicate across different experiments, particularly noted as diagonal lines running from the upper left to the lower center in images b, g left, and h. Could you clarify the origin of these identical stains? If these are artifacts and the original fluorescent images are clear, I would recommend substituting the current images with the unblemished versions.
(2) Regarding Figure 3, in some lanes, the same locations as the PCR products in template 23 appear distinctly whiter than the background. Quantifying the fluorescence intensity in this region would be essential to assess the stopping efficiency accurately. Could you provide an explanation or further clarification on this observation?

Response_1
Thank you for your valuable feedback regarding the gel images. We greatly appreciate your point. In response to your first comment (1), we have identified that the presence of scratches on the gel imaging equipment's plate caused the common lines seen in the gel images of Figure 3b, g, and h. Additionally, regarding your second comment (2), we have determined that the reason for the ODN_23 band's position appearing whiter than the background was due to the dye present in the loading buffer. The problematic gel matches the one highlighted in your first comment, specifically Figure 3b, g, h.
To address both comments (1) and (2), we have re-conducted the primer extension assay for Figure 3b, g, h. During this process, we used a loading buffer without dye. Furthermore, we ensured that the gel was photographed using a clean plate to eliminate any issues. As a result, we have updated Figure 3b, g, h with new gel images and re-evaluated the chain extension termination efficiency.
Additionally, since there were changes in the results of the chain extension termination efficiency due to these adjustments, we have also made modifications to the following two sentences in the main text regarding the results obtained when using ODN_2 and ODN_28.

“The product that stopped at the modification position was the highest at 90% with Phusion High Fidelity (Fig. 3b).”
→“The product that stopped at the modification position was the highest at 78% with Deep Vent (Fig. 3b).”

“ODN_28, which introduced compound 18 with a protecting group in both the base and phosphate moieties, gave a slight extension-stopped product of approximately 27% when using Pfu and Deep Vent.”
→“ODN_28, which introduced compound 18 with a protecting group in both the base and phosphate moieties, gave a slight extension-stopped product of approximately 25-30% when using Pfu, Q5 high fidelity, and Deep Vent.”

Comment_2
(3) In Table 1, the listed sequence for ODN_25 appears to contain an additional 'C' compared to its description in Figure 3. Would you mind verifying this sequence and updating the table accordingly to ensure accuracy?

Response_2
Thank you for pointing out the error in the nucleotide sequence of ODN_25 in the table. We appreciate your attention to detail. As you correctly noted, there was an extra C nucleotide in the sequence. We have replaced Table 1 with the corrected nucleotide sequence for ODN_25. We apologize for any confusion caused by the error.

Comment_3
(4) While you have compared the DNA linkage efficiency to that of the restriction enzyme method, it might be beneficial to also compare it to other methods like In-Fusion cloning, which improves ligation efficiency through the addition of extended adhesive ends via PCR. Including such a comparison could significantly underscore the unique advantages of your method.

Response_3
Regarding the efficiency of DNA ligation, we appreciate the suggestion to emphasize the advantages of our method through comparisons with techniques other than restriction enzymes. It is worth noting that methods such as Gibson assembly and In Fusion cloning, which rely on DNA fragments containing homologous sequences for ligation, are primarily applicable to circular DNA synthesis. We tried to evaluate the ligation efficacy of Gibson assembly using 5' FAM-labeled DNA fragments. However, the assembly uses 5' exonuclease to generate homologous sticky ends, and it removes the label. Furthermore, digestion lasts until a circular DNA is formed, making DNA fragment bands smear. These features make estimating ligation efficacy by electrophoretic mobility shift assay challenging. Our approach utilizing stop primers for DNA ligation offers versatility by allowing both circular and linear DNA constructs. Therefore, in this study, we undertook the challenge of constructing long linear DNA fragments derived from λ phage DNA, showcasing the broad applicability of our method beyond circular DNA constructs. We have incorporated these points into the main text as follows: [In addition to the restriction enzyme method used for comparison in this study, there are existing DNA ligation methods such as Gibson assembly21,22 and In-Fusion cloning.48 However, these techniques are limited to the synthesis of circular DNA. In contrast, DNA ligation using stop primers can be applied to the ligation of linear DNA as well, offering a broader range of applications.].

Referee: 3
Thank you very much for dedicating your valuable time and effort to reviewing our manuscript. We sincerely appreciate your favorable evaluation of our method for synthesizing DNA fragments with sticky ends. Your comments were exceptionally valuable, and as outlined in our responses, we have incorporated additional experimental data and made necessary revisions to improve the quality of the manuscript. The changed or added parts are highlighted in yellow. We hope that these revisions meet your expectations and that the revised manuscript now meets your satisfaction. Your feedback has been invaluable to us, and we are grateful for your contribution to enhancing our work.

Comment_1
(1) The decaging product analyses in Figure 2 are based on only one technique, HPLC retention time. These data alone can only prove that there are some chemical changes occurred after light irradiation. I recommend ESI-MS analyses on the decaging products (those in Figure 2b and 2c) to confirm they are indeed the desired products, excluding the possibilty of incomplete decaging or any photo-induced damage to the DNA products. In addition, I recommend the authors to provide their ESI-MS data chart (images), instead of only numbers in tables.

Response_1
Thank you for your valuable feedback regarding the need for confirmation of the identity of DNA products following photo-irradiation. We appreciate your suggestion. To address this concern, we conducted ESI-MS analysis on the ODN_1-4, 6, and 7 samples subjected to photo-irradiation in Figure 2b and c. The results of our analysis confirmed that no incomplete decaging products or products damaged by light were observed, indicating that only the desired products were obtained. We have included this information in the main text as follows: [Upon conducting mass analysis of the products after light irradiation using Electrospray Ionization Mass Spectrometry (ESI-MS), no products derived from DNA damage were observed. Only the molecular weight of the target was detected (Fig. S1).].

Additionally, we have added a new section to the Supporting Information titled 'Evaluation of removal of o-nitrobenzyl groups by photo irradiation' to provide a detailed account of the experimental methodology. The data chart from the ESI-MS analysis has been included as Figure S1. We hope these additions provide a clearer understanding of the quality and identity of the DNA products following photo-irradiation.

Comment_2
(2) Because 365 nm UV light is applied in the method, there is possibility of photo-induced damages to the DNA, and the consequence may be more servere (such as wrong protein translation) when the DNA fragments are long. I recommend the authors using either the plasmid (Figure 5a) or the long DNA product (Figure 5b) to do a Sanger sequencing analysis, to show that the product sequence is indeed very clean.

Response_2
Thank you for your feedback regarding the sequence analysis experiments of the ligation products. We appreciate your attention to this aspect of our research. In response to your comment, we selected 16 blue colonies obtained in Figure 5a, conducted culturing and plasmid extraction, and subsequently performed sequence analysis using the Sanger method. The results of this analysis revealed that out of the 32 junction sites, 3 locations exhibited mutations. These observed mutations included instances of mismatch mutations during gap-filling by the polymerase during ligation, as well as cases where bases were skipped. Besides the mutations on junction sites, there are no mutations around the junction site within 725 bp. Thus, the 365 nm photoirradiation did not induce DNA mutations. It is important to note that in the majority of cases, we confirmed the successful construction of the plasmids with the correct sequences.
We have incorporated this information into the main text as follows: [Sixteen blue colonies were picked and cultured, and plasmids were extracted from them. The sequences of the obtained plasmids were analysed using the Sanger method. The results revealed that mutations occurred at three of the 32 ligation sites (Fig. S3). The observed mutations were mismatch mutations that occurred during gap-filling by the polymerase, or base skipping. However, the construction of plasmids with the correct sequence was confirmed in most of the samples. In addition, no mutations were observed in regions other than the ligation site. Thus, the 365 nm photoirradiation used to remove the o-nitrobenzyl modification did not induce DNA mutations.]. Additionally, we have added a new section to the Supporting Information titled 'Sequence analysis of constructed plasmid' to provide a detailed account of the experimental methodology. The results of the sequence analysis have been included as Figure S3. We hope that these additions enhance the understanding of the sequence analysis results and their implications.

19-Feb-2024

Dear Dr Abe:

Manuscript ID: CB-ART-10-2023-000212.R1
TITLE: Development of PCR Primer Enabling the Design of Flexible Sticky Ends for Efficient Concatenation of Long DNA Fragments

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Reviewer 3

The authors have addressed my comments to the original manuscript in the revised version. I recommend its acceptance for publication.

Reviewer 2

All of the reviewers' comments have been addressed appropriately. I thank the authors for their diligent efforts and revisions to the manuscript. The addition of new experiments and descriptions has greatly improved the manuscript. I strongly recommend the publication of the revised manuscript without further changes.