PONE-D-24-28006Peptide Nucleic Acids can form hairpins and bind RNA-binding proteinsPLOS ONE

Dear Dr. Kwan,

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.

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As you will see one Reviewer suggested minor revisions, while the other one suggested a major revision. However, based on the comments from both Reviewers I think that a minor revision is the most appropriate choice. Reviewer 1, who is an expert in RNA-ligand and protein-ligand interaction, as well as in structural biology, is extremely satisfied with the set of experiments used to prove most of your claims. Nonetheless, I will ask you to correct the manuscript by addressing all of their concerns:

1. Add a discussion of the future directions of how to improve the PNA/protein binding strength and specificity. In particular, what kind of PNA SL modifications: structural, chemical, sequence (?) could make it a better competitor for the cognate RNA SL?

2. It is not clear what role glutamate has in binding, if any at all. Please explain it.

3. What is the OO linker?

4. Fig 2A: why are the three black lines different in the three panels?

5. Line 472: It is not clear what the authors mean by “using GDP binding as a positive control”. Do they mean competitive inhibitor?

6. The RNA-FtsY interaction is well-known to be a transient interaction. Does the PNA have any meaningful inhibitory role in a functional SRP assay (GTP hydrolysis, protein targeting, secretion etc?)

Reviewer 2 also suggested to compare one PNA without glutamate to assess this. I would strongly encourage you to perform this experiment; however, if not possible please justify it. Also, I would ask you to address to the best of your abilities if Ffh-RNA binding can be similarly inhibited by the synthesized PNA. It would be ideal to have experimental data to support this answer.

Al the best,

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Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear Prof. Kwan,

I want to thank your for your submission. As you will see one Reviewer suggested minor revisions, while the other one suggested a major revision. However, based on the comments from both Reviewers I think that a minor revision is the most appropriate choice. Reviewer 1, who is an expert in RNA-ligand and protein-ligand interaction, as well as in structural biology, is extremely satisfied with the set of experiments used to prove most of your claims. Nonetheless, I will ask you to correct the manuscript by addressing all of their concerns:

1. Add a discussion of the future directions of how to improve the PNA/protein binding strength and specificity. In particular, what kind of PNA SL modifications: structural, chemical, sequence (?) could make it a better competitor for the cognate RNA SL?

2. It is not clear what role glutamate has in binding, if any at all. Please explain it.

3. What is the OO linker?

4. Fig 2A: why are the three black lines different in the three panels?

5. Line 472: It is not clear what the authors mean by “using GDP binding as a positive control”. Do they mean competitive inhibitor?

6. The RNA-FtsY interaction is well-known to be a transient interaction. Does the PNA have any meaningful inhibitory role in a functional SRP assay (GTP hydrolysis, protein targeting, secretion etc?)

Reviewer 2 also suggested to compare one PNA without glutamate to assess this. I would strongly encourage you to perform this experiment; however, if not possible please justify it. Also, I would ask you to address to the best of your abilities if Ffh-RNA binding can be similarly inhibited by the synthesized PNA. It would be ideal to have experimental data to support this answer.

Al the best,

Prof. Mauricio Comas-Garcia

Handling Editor

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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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: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Review in the manuscript “Peptide Nucleic Acids can form hairpins and bind RNA-binding proteins” by Yichen Zhong et.al.

In this manuscript the authors explore an innovative idea that the peptide nucleic acids (PNAs) can be used as a drug that will compete with the RNA for its functional partner protein binding. They hypothesize that the short PNA molecule of the same sequence as prototype RNA can fold into the same secondary structure and bind the same protein partner via the same interface. This is far from obvious, and the authors managed to prove this hypothesis for the short stem loop (SL) structure by using the impressive array of experimental approaches, including NMR, SPR, CD, EMSA, and MST. This work is convincing as a proof of principle. The authors have shown quite convincingly by a combination of approaches that their PNA SL folds in the same hairpin structure as analogous RNA and binds protein with the same surface. The big advantage of the PNA vs RNA drug is that PNA is not degradable by the cellular nucleases.

However, the efficiency of such PNA mimic of RNA SL in competing for its partner protein at least in the case studied by authors is relatively low. Thus, the competition binding experiments presented in Fig. 2 suggest that even the best competitor PNAtelL of the RNA SL for binding the partner protein I only weakly effective. Indeed, addition of 5 �M of this competitor PNA SL to the protein makes the cognate RNA SL bind to it only ~2.5-fold weaker, with Kd changing from ~100nM to ~300nM. Based on that result we can roughly estimate that the PNAtelL Kd for that protein is ~50-fold weaker that the RNA Kd for the same protein. However, maybe the PNA can be further modified to improve its cognate protein binding. This seems possible, as according to the same competition studies, the strength of PNA/protein interaction is strongly affected by the length of the PNA stem, the sequence of its loop, type of the PNA or modified RNA backbone used. I believe this paper would benefit from discussion of the future directions of how to improve the PNA/protein binding strength and specificity. What kind of PNA SL modifications: structural, chemical, sequence (?) could make it a better competitor for the cognate RNA SL?

Reviewer #2: This communication by Kwan and coworkers reports the use of peptide nucleic acids (PNAs) as RNA mimics for competitive inhibition of RNA-protein interactions. The authors have demonstrated this with the help of two examples, including that of SRP inhibition.

Overall, the text is well written and is worthy of publication. However, there are a few queries that need to be carefully addressed before the paper can be considered suitable for publication.

It is not clear what role glutamate has in binding, if any at all. The authors should compare one PNA without glutamate to assess this.

What is OO linker?

Fig 2A: why are the three black lines different in the three panels? I understand that they are all measuring RNA-FtsY binding in the absence of PNA. As Ffh is also well-known to bind to RNA, the authors must check whether Ffh-RNA binding can be similarly inhibited by the synthesized PNA.

Lin2 472: It is not clear what the authors mean by “using GDP binding as a positive control”. Do they mean competitive inhibitor?

The RNA-FtsY interaction is well-known to be a transient interaction. Does the PNA have any meaningful inhibitory role in a functional SRP assay (GTP hydrolysis, protein targeting, secretion etc?)

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Reviewer #1: Yes: Ioulia Rouzina

Reviewer #2: No

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Peptide Nucleic Acids can form hairpins and bind RNA-binding proteins

PONE-D-24-28006R1

Dear Dr. Kwan,

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.

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Kind regards,

Mauricio Comas-Garcia

Academic Editor

PLOS ONE