A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies

Yi Zhang; Takuro Nunoura; Daisuke Nishiura; Miho Hirai; Shigeru Shimamura; Kanako Kurosawa; Chieko Ishiwata; Shigeru Deguchi

doi:10.1371/journal.pone.0244464

Peer Review History

Original SubmissionOctober 5, 2020
28 Oct 2020 Decision Letter - Ruslan Kalendar, Editor PONE-D-20-31271 A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies PLOS ONE Dear Dr. Zhang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Dec 12 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Ruslan Kalendar, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have a patent relating to material pertinent to this article. 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Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Reviewer #1: Zhang and colleagues present a fascinating technique using a femtoliter array for investigating DNA-cleaving enzymes. It would certainly be of interest if this microfabricated array could be mass produced as not all of us have access to clean-room manufacturing. The actual technique for making and usinfg the array really needs a figure of its own to make the process clear. They find that the restriction enzymes investigated do not give full cleavage as assessed by cell free protein synthesis. It is a pity that more detailed kinetic studies were not performed. It is essential that the enzyme concentrations are clearly stated - using "activity units" is not satisfactory as I am sure that some of these enzymes will have concentrations in excess of the DNA concentration in the digest thus the assays will be in a single-turnover regime while others may not be. (New England Biolabs do have this sort of information if asked) The lack of full cleavage by restriction enzymes is well known hence the development of the HF variants. It is also well known that the sequence surrounding the target site has an influence on cleavage efficiency - this should be investigated by analysing their template sequence. The template sequences for the Scarlet and Turquoise genes need to be added to figure S1. Figure S5 has a typographical error - it should have nM/min rather than nm/min. Reviewer #2: Zhang et al., present a high sensitivity, facile and inexpensive method of assessing DNA digest or degradation efficiency. They use an elegant digital cell free protein synthesis assay as a read out of digest completeness for a variety of DNA restriction and DNA degrading enzymes and demonstrate for this system a low limit of detection. The system features a PCR free method without the need for heat inactivation or expensive instrumentation. The manuscript is well written, the figures well designed and the work well done. I have only a few minor amendments, listed below that should improve the clarity of the presentation. General comments: I would argue that the manuscript should feature one additional data figure in the main body of the text that compares the electrophoresis (currently shown in SFig 9) vs. the digital cell free protein synthesis results shown in tabular form. This would present data of interest to many general readers in a non-textual manner and without the need to access the supplementary information. In the last paragraph of the discussion a single high efficiency digest of a type IIS RE is used to generalize that consensus that this class of RE requires two copies may be incorrect. This conclusion seems unjustified based on the results shown. Minor edits: -p21 line 326 Michalis-Menton should be Michaelis-Menton -p24 line 376 ‘a single band on both of CE chips..’ should read‘a single band on both CE ’ chips…’ -p29 line 493 laboratorial should read as the simpler laboratory -p30 line 524 ‘may greatly exceed our thought’ should read ‘may greatly exceed our expectations’ -p31 line 533 ‘As a consequence, archaea and bacteria prefer or have to coexist…’ the intended meaning of this sentence is very unclear. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No https://doi.org/10.1371/journal.pone.0244464.r001
Revision 1
8 Dec 2020 Author Response Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: We prepared the manuscript and named each file according to the provided style templates. 2. We note that you have a patent relating to material pertinent to this article. Please provide an amended statement of Competing Interests to declare this patent (with details including name and number), along with any other relevant declarations relating to employment, consultancy, patents, products in development or modified products etc. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. This information should be included in your cover letter; we will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests Response: We added the patent name and application number and included the statement “This does not alter our adherence to PLOS ONE policies on sharing data and materials” in the amended statement of Competing Interests. Now, the statement is: I have read the journal's policy and the authors of this manuscript have the following competing interests: [YZ and TN are inventors on a pending patent (Name: methods of obtaining the digestion efficiency of nucleases; Application number: 2019-232639) related to this work.] This does not alter our adherence to PLOS ONE policies on sharing data and materials. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. Response: We provided the requested original images in a new supporting information file named as “S1_raw_images”. Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Yes Reviewer #2: Yes 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes 3. Have the authors made all data underlying the findings in their manuscript fully available? Reviewer #1: Yes Reviewer #2: Yes 4. Is the manuscript presented in an intelligible fashion and written in standard English? Reviewer #1: Yes Reviewer #2: Yes 5. Review Comments to the Author Reviewer #1: Zhang and colleagues present a fascinating technique using a femtoliter array for investigating DNA-cleaving enzymes. It would certainly be of interest if this microfabricated array could be mass produced as not all of us have access to clean-room manufacturing. Response: We thank the reviewer for the positive evaluation of our work and the interest in our array device. We are seeking funding to upgrade the microfabrication process and realize mass production. The actual technique for making and usinfg the array really needs a figure of its own to make the process clear. Response: We modified Fig. 1B to make the experiment process clear. They find that the restriction enzymes investigated do not give full cleavage as assessed by cell free protein synthesis. It is a pity that more detailed kinetic studies were not performed. It is essential that the enzyme concentrations are clearly stated - using "activity units" is not satisfactory as I am sure that some of these enzymes will have concentrations in excess of the DNA concentration in the digest thus the assays will be in a single-turnover regime while others may not be. (New England Biolabs do have this sort of information if asked) The lack of full cleavage by restriction enzymes is well known hence the development of the HF variants. It is also well known that the sequence surrounding the target site has an influence on cleavage efficiency - this should be investigated by analysing their template sequence. Response: This comment consists of two aspects. One is about the enzyme kinetics study, and the other one is about the investigation of sequence-dependent cleavage efficiency. For the first one, we got the concentration information (as shown in a new S1 Table in the Supporting Information) from NEB and showed the applicability of our method for the enzyme kinetics measurement. The result was summarized in a new sub-section entitled “Simplified measurement of enzyme kinetics based on digital counting”. For the second one, we modified the sequence surrounding the target site and confirmed the influence on cleavage efficiency using the digital counting method. The result was added to the sub-section “Comprehensive analyses of restriction digest efficiencies”. Accordingly, we updated the Acknowledgments section and added some words to the Materials and Methods section for the above contents. Based on the newly gained insight, we also added some words to the Discussion section. The template sequences for the Scarlet and Turquoise genes need to be added to figure S1. Response: We added the template sequences of mScarlet and mTurquoise2 genes to Figure S1 and revised the figure caption accordingly. Figure S5 has a typographical error - it should have nM/min rather than nm/min. Response: This typographical error in Figure S5 was corrected. Reviewer #2: Zhang et al., present a high sensitivity, facile and inexpensive method of assessing DNA digest or degradation efficiency. They use an elegant digital cell free protein synthesis assay as a read out of digest completeness for a variety of DNA restriction and DNA degrading enzymes and demonstrate for this system a low limit of detection. The system features a PCR free method without the need for heat inactivation or expensive instrumentation. The manuscript is well written, the figures well designed and the work well done. I have only a few minor amendments, listed below that should improve the clarity of the presentation. Response: We appreciate the reviewer for the very positive evaluation of our work. General comments: I would argue that the manuscript should feature one additional data figure in the main body of the text that compares the electrophoresis (currently shown in SFig 9) vs. the digital cell free protein synthesis results shown in tabular form. This would present data of interest to many general readers in a non-textual manner and without the need to access the supplementary information. Response: We offered a new Fig. 3 in the main text that combined the electrophoresis results and the digital counting results (i.e., the digest efficiency data). I guess that the “SFig 9” in the reviewer’s comment should be read as “S7 Fig”, since the tabular form does not contain the digital counting results of S9 Fig. The new Fig. 3 presented the data “to general readers in a non-textual manner and without the need to access the supplementary information”. In the last paragraph of the discussion a single high efficiency digest of a type IIS RE is used to generalize that consensus that this class of RE requires two copies may be incorrect. This conclusion seems unjustified based on the results shown. Response: We modified the sentence to make the argument more precise than before. Minor edits: -p21 line 326 Michalis-Menton should be Michaelis-Menton Response: The typo was corrected. -p24 line 376 ‘a single band on both of CE chips..’ should read‘a single band on both CE ’ chips…’ Response: The sentence was revised. -p29 line 493 laboratorial should read as the simpler laboratory Response: The word was revised. -p30 line 524 ‘may greatly exceed our thought’ should read ‘may greatly exceed our expectations’ Response: The word was revised. -p31 line 533 ‘As a consequence, archaea and bacteria prefer or have to coexist…’ the intended meaning of this sentence is very unclear. Response: The sentence was improved to make the meaning clear. A marked-up copy of our manuscript that highlighted changes made to the original version was upload as a separate file labeled “Revised Manuscript with Track Changes”. An unmarked version of our revised paper without tracked changes was upload as a separate file labeled “Manuscript”. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0244464.r002
10 Dec 2020 Decision Letter - Ruslan Kalendar, Editor A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies PONE-D-20-31271R1 Dear Dr. Zhang, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ruslan Kalendar, PhD Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244464.r003
Formally Accepted
22 Dec 2020 Acceptance Letter - Ruslan Kalendar, Editor PONE-D-20-31271R1 A single-molecule counting approach for convenient and ultrasensitive measurement of restriction digest efficiencies Dear Dr. Zhang: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Prof. Ruslan Kalendar Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0244464.r004

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