PONE-D-20-27312

A New Class of Polymeric Fluorescent Dyes Assembled Using

A DNA Synthesizer

PLOS ONE

Dear Dr. Matray,

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.

Ïn particular, the authors should make sure to address the following points:

a) Increasing the resolution and quality of images of all figures has been brought up by both reviewers, so this point should be addressed before acceptance.

b) The synthesis portion should include further characterization details. The authors should also indicate a possible path towards synthesis in larger quantities to cater to the envisioned applications (although it is not necessary that they carry out a scale up experimentally at this time). It may also be considered to refer to the materials obtained as oligomers rather than polymers, if the degree of polymerization is relatively low. 

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Marco Bonizzoni, Ph.D.

Academic Editor

PLOS ONE

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[All work funded by Sony Corporation and Sony Imaging Products & Solutions Inc.  

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper demonstrates the applicability of a DNA synthesizer for the synthesis of fluorescently-labeled phosphodiester oligomers. The authors successfully prepared a series of oligomers covalently tagged by two fluorescent dyes at the both ends, and discussed the effects of oligomer length and type on quenching of the dyes. This paper reports new oligomers, and thus the authors should provide a summary table of LC-MS results, including expected and experimental molecular weights, purity, yield, for all of the oligomers. They should also provide characterization data for the post-functionalization of polyamines with fluorophores, possibly obtained using LC-MS (and NMR). In addition, the following points should be considered for publication of this work in PLOS ONE.

1. The title should be changed so that it represents the achievements of this work, which I think is the application of a DNA synthesizer for synthesis of various oligomers. This paper does not report any new class of fluorescent dyes, as the fluorescence of the oligomers originates from conventional fluorescent dyes, but not from the oligomer chains.

2. The authors are strongly encouraged to quantitatively analyze the relationship between the fluorophore distance, which may be estimated computationally, and quenching efficiency. The quenching efficiency is well-known to be controlled by distance between the fluorophores and also their mutual orientation. This analysis should help the authors discuss the roles of linker properties in quenching more clearly.

3. In Introduction, the authors should discuss similar quenching studies using other oligomeric linkers like oligonucleotides, peptides and synthetic polymers to highlight the novelty of this work based on phosphodiester oligomers. For protein labeling (Line 50), the authors should also mention genetically encoded fluorescent proteins and other materials such as conducting polymer dots.

4. The authors should consider a possibility of the complexation of PEG with Na+ for the modeling in Figure 7.

5. Improve the quality of the figures. The resolution of many graphs should be higher than 300 dpi. The labels on the x- and/or y-axes are missing for Figures 1A, 4BC and 9. The style of the graphs in Figure 9 should be the same, and Figure 9D should be replaced as the fluorescence intensity is saturated for 10X. Spectra should be given in Figure SM1 instead of the plots. All the captions should provide experimental conditions such as excitation wavelength, concentrations, solution conditions and HEG/PEG (for Figure 9).

Finally, the authors are strongly encouraged to edit the paper more carefully. There are a number of typos (for example, “phosphoramdite” on Line 99) and grammatical issues (for example, “correlate” on Line 198 and “total” on Line 214). Define Gen I, II and III clearly. The synthesized materials sound oligomers, rather than polymers, considering their relatively small polymerization degrees.

Reviewer #2: The manuscript entitled 'A new class of polymeric fluorescent dyes assembled using

5 a DNA synthesizer' submitted by Yada et al describes a novel and facile synthetic method to obtain multivalent (polymeric) fluorescent dye with optimizable properties. Overall, it is a well-written manuscript and sufficient amount of experiments (along controls) have been done. The paper is acceptable for PLOS One with minor revision. These revisions will be needed, in my opinion, in following two areas:

a) Increasing the resolution and quality of images of all figures, particularly fluorescent spectroscopic plots. Apparently, the legends are unreadable, and in others axis-associated fonts are too small.

b) The synthesis is applicable, in its current format, in microscale. A paragraph regarding scalability of the process is requested.

c) Since, the manuscript is aimed for PLOS One, the audience of which has diversified background, a more understandable reaction scheme will improve the impact of the manuscript.

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Reviewer #1: No

Reviewer #2: No

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A Novel Class of Polymeric Fluorescent Dyes Assembled Using

A DNA Synthesizer

PONE-D-20-27312R1

Dear Dr. Matray,

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.

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Kind regards,

Marco Bonizzoni, Ph.D.

Academic Editor

PLOS ONE