From the journal RSC Chemical Biology Peer review history

28-Jul-2023

Dear Dr BRASELMANN:

Manuscript ID: CB-COM-06-2023-000099
TITLE: Evaluating Riboglow-FLIM probes for RNA sensing

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

After careful evaluation of the manuscript and reviewers’ comments, I regret to inform you that I do not find your manuscript suitable for publication due to the limited novelty and scope of the results presented. Both reviewers find that additional data are needed to support the conclusions drawn in your manuscript. For these reasons, I regret that your manuscript has been rejected in its current form.

However, if you are able to fully address the concerns raised by the reviewers in the reports below, I will consider a substantially revised manuscript with additional experimental data that address all of the reviewers’ comments and recommendations. If you choose to resubmit your manuscript, please include a point by point response to the reviewers’ comments and highlight the changes you have made.

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Yours sincerely,
Claudia Höbartner
Associate Editor, RSC Chemical Biology

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Reviewer 1

Nadia Sarfraz et al. in the submitted manuscript “Evaluating Riboglow-FLIM probes for RNA sensing” report further characterization of Riboglow tag and its applications for FLIM imaging. The authors have further investigated properties of three Riboglow ligands: Cbl-4xGly-ATTO 590, Cbl-5xPeg-ATTO 590, and Cbl-Cy5. The authors have determined fluorescence lifetime changes after binding to RNA A-tag variant of Riboglow. In addition, the authors have demonstrated that fluorescence lifetime of the probes is dependent of the cell type by using FLIM.

Overall, this well written manuscript is reporting a valuable characterization of Riboglow ligands. Thus, I am happy to recommend this manuscript to be accepted for publishing in RSC Chemical Biology after a major revision because some additional experiments and explanations are needed:

Major point:
1. The authors evaluate only three Riboglow ligands: Cbl-4xGly-ATTO 590, Cbl-5xPeg-ATTO 590, and Cbl-Cy5. These ligands are could give a good systematic overview on how Riboglow is influencing fluorescence lifetime or structure-function relation under different cellular environment. I propose to make the study more systematic by testing a wider range of PEG linkers involving single PEG, 3xPEG and 9xPEG. Furthermore, I suggest to use only one fluorophore (ATTO 590) for simplicity.
2. The authors make strong statement about differences in cell lines based on two cell lines (HOS and U-2 OS). This is clearly not sufficient, I would be interesting to test non-cancer cell line (fibroblasts) or stem cells. This would strengthen the proposed observation and extend the scope of the paper.
3. The authors are not evaluating the stability of reported probes (Cbl-4xGly-ATTO 590, Cbl-5xPeg-ATTO 590, and Cbl-Cy5) in the reported cell types. It could be that the metabolic stability is leading to the differences in fluorescence lifetime. Please test this.

Minor points:
1. Please comment how efficient is bead loading of the probes. It would be useful to provide the percentage of loaded cells vs not labelled cells.
2. Please comment what are the differences between HOS and U-2 OS cell lines in terms of metabolic activity and gene expression level. It is not clear if these cells lines differ only in the shape or more differences are involved which might affect FILM results.
3. The authors are providing an obvious general statement “Prior to each application, it is essential to systematically assess and optimize each experimental variable to validate accurate and reliable fluorescence lifetime measurements, ensuring obtained results reflect the molecular events of interest and provide meaningful insights.” I suggest to make it more specific to Riboglow system, so reader can benefit from the information presented in the paper.

Reviewer 2

Sarfraz et al. have recently introduced a new RNA detection platform called Riboglow-FLIM, which relies on the fluorescence lifetime changes of fluorogenic probes (such as Cy5-Cbl and Atto590-Cbl) when bound to the Riboglow tag. In their manuscript, the authors investigate the impact of various factors, including the nature of fluorophore (Cy5 and Atto590), linker length between the fluorophore and Cbl, and cell type (HOS and U2OS) on the fluorescence lifetime of the probes.

I find the novelty of this study relatively low. However, Riboglow-FLIM is a new technique and I agree with the authors that a comprehensive study to understand the potential benefits and limitations of Riboglow-FLIM compared to existing techniques is necessary. One criticism of the manuscript is the limited scope of the study, with only two fluorophores and two cell lines being investigated. Additionally, the conclusions drawn about the linker length between the fluorophore and Cbl are not clear.
Furthermore, some results presented in the manuscript, such as Kd values, certain lifetimes, and cell viability data presented on page 7, seem to have been previously reported and only replicated in this study.

Most importantly, it is disappointing that there is no single RNA imaging experiment performed in live cells using Riboglow-FLIM.

In summary, while Sarfraz et al.'s work on Riboglow-FLIM is noteworthy, the manuscript may benefit from addressing the mentioned limitations and expanding the scope of the study to provide a more comprehensive evaluation of the technique's potential.

Additional comment:

Suppl. Fig.2B: What is the stoichiometry between Cbl and the aptamer? The data do not seem to fit 1:1 binding model.

03-Dec-2023

Dear Dr BRASELMANN:

Manuscript ID: CB-COM-10-2023-000197
TITLE: Evaluating Riboglow-FLIM probes for RNA sensing

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to the previous reviewers and additional reviewers and I have now received their reports which are copied below.

After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to consider your manuscript for publication after revisions.

Please revise your manuscript to fully address the reviewers’ comments. When you submit your revised manuscript please include a point by point response to the reviewers’ comments and highlight the changes you have made. Full details of the files you need to submit are listed at the end of this email.

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I look forward to receiving your revised manuscript.

Yours sincerely,
Claudia Höbartner
Associate Editor, RSC Chemical Biology

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

Reviewer 1

Nadia Sarfraz et al. in the re-submitted manuscript “Evaluating Riboglow-FLIM probes for RNA sensing” have substantially improved the quality of the reported research and satisfactory addressed issues pointed by reviewers. This work is suitable for commination format because it is high quality and significance, and of high interest to the chemical biology community. Thus, I am happy to recommend this manuscript to be accepted for publishing in RSC Chemical Biology after a minor revision because one important point need to be addressed before publication:

In the Supplementary Table 4, the authors list P-values equal to “zero” (p = 0.00000). This is fundamentally not correct since such values could be obtained only in ideal experiments (simulations, not measurements). Please provide correct P-values and check the calculations.

Reviewer 3

The authors previously developed Riboglow-FLIM, a technique for tagging and tracking RNA in live cells using a fluorescence lifetime-measuring probe. The current study evaluated the effects of varying the linker and changing the environments on fluorescence lifetime. The experiments included both in vitro probe variants and in vivo applications across multiple mammalian cell types, using fluorescence lifetime imaging microscopy (FLIM) and assessing any impact on cell health. While the results can be useful for choosing the right probe and considering the cellular environment in Riboglow-FLIM's application for RNA detection, the following deficiencies should be addressed.

1. Riboglow-FLIM was developed to visualize engineered RNA species in cells. However, the current study is limited to the dyes’ characterization in parental cells which do not express the target RNA if this reviewer’s understanding is correct. While investigation of such “background” signals can be useful, a direct comparison between such “background” to signal (in presence of target RNA species) would be critical for understanding the differences between the two. It is recommended the authors perform measurement on cells expressing RNA target of Riboglow-FLIM and discuss the differences.
2. If the above understanding (this study being limited to the dyes’ characterization in parental cells which do not express the target RNA) is correct, where does the “background” come from in parental cells is an important question that the manuscript fails to discuss. Does the background come mostly from the free dye, or the dye bound to certain cellular targets (proteins or nucleic acids)? Any experiments or data to illuminate on this?

We appreciate the opportunity to submit a revised manuscript and address reviewer comments.

Below is a point-by-point response to comments by the editor and the reviewers. To address specific reviewer comments, key text that addresses the comments is marked in yellow in the main file.


REVIEWER REPORT(S):
Referee: 1

Comments to the Author
Nadia Sarfraz et al. in the re-submitted manuscript “Evaluating Riboglow-FLIM probes for RNA sensing” have substantially improved the quality of the reported research and satisfactory addressed issues pointed by reviewers. This work is suitable for commination format because it is high quality and significance, and of high interest to the chemical biology community. Thus, I am happy to recommend this manuscript to be accepted for publishing in RSC Chemical Biology after a minor revision because one important point need to be addressed before publication:

RESPONSE:
We appreciate the overall positive assessment and agree that the manuscript has substantially improved.

In the Supplementary Table 4, the authors list P-values equal to “zero” (p = 0.00000). This is fundamentally not correct since such values could be obtained only in ideal experiments (simulations, not measurements). Please provide correct P-values and check the calculations.

RESPONSE:
We thank the reviewer for this very important note. This was an oversight of a typo, and it should read ****p ≤ 0.0001. The table has been updated appropriately.

Referee: 3

Comments to the Author
The authors previously developed Riboglow-FLIM, a technique for tagging and tracking RNA in live cells using a fluorescence lifetime-measuring probe. The current study evaluated the effects of varying the linker and changing the environments on fluorescence lifetime. The experiments included both in vitro probe variants and in vivo applications across multiple mammalian cell types, using fluorescence lifetime imaging microscopy (FLIM) and assessing any impact on cell health.

RESPONSE:
We are grateful for the positive note about the foundational relevance of this study. We envision that this detailed study with important quantitative controls lays the foundation for future applications, together with the proof-of-principle studies we recently published elsewhere. Please note that one of the publications was still in review when the first submission of this manuscript was in review with RSC Chemical Biology as well. We updated these references and important conclusions more clearly throughout (Sarfraz et al, 2023, Nat. Comm; Sarfraz et al, 2023, Biophys. Rep.).

While the results can be useful for choosing the right probe and considering the cellular environment in Riboglow-FLIM's application for RNA detection, the following deficiencies should be addressed.

1. Riboglow-FLIM was developed to visualize engineered RNA species in cells. However, the current study is limited to the dyes’ characterization in parental cells which do not express the target RNA if this reviewer’s understanding is correct.

RESPONSE:
The reviewer understands correctly that the current study is focusing on the probe(s) in different parental cells. We demonstrated the overall change in FLIM signal in the presence of RNA in live cells elsewhere as proof-of-concept (Sarfraz et al, 2023, Nat. Comm.; Sarfraz et. al., 2023, Biophys. Rep.). While we are encouraged by these pilot observations, a systematic and exhaustive assessment of cell health across diverse cell systems has been lacking so far – this has been correctly pointed out in the previous review of this work and is hence emphasized here. As pointed out before, this methodical and exhaustive evaluation of cellular conditions is critical for further tool development efforts and applications. Additionally, we include systematic and methodical evaluations of FLIM in vitro here to bridge the very complex cellular proof-of-concept observations and systematic evaluations of conditions in the test tube (in the presence / absence of the RNA ligand). We made this point more clearly in the main text.

While investigation of such “background” signals can be useful, a direct comparison between such “background” to signal (in presence of target RNA species) would be critical for understanding the differences between the two. It is recommended the authors perform measurement on cells expressing RNA target of Riboglow-FLIM and discuss the differences.

RESPONSE:
Thank you to the reviewer for this important point. We re-organized Figure 2 to make the point more clearly that we did perform the experiment of adding the RNA to the purified system. We would like to point out that we did perform the suggested experiment (probe in cells producing the RNA) in previous pilot studies (Sarfraz et al, Nat. Comm, 2023; Sarfraz et al. Biophys. Rep., 2023). We acknowledge that not all of this was published during the review of this current manuscript. The now published live cell data were added to Supplementary Table 3 for a clearer side-by-side comparison of all Riboglow-FLIM data. The published proof-of-concept studies demonstrate that the fluorescence lifetime significantly changes in the presence of the RNA in select live cell examples, in line with in vitro measurements (see Supplementary Table 3 for a comprehensive summary). Before expanding on our anecdotal observations in cells, we chose to carefully and systematically assess different environments in vitro and in cells for this study as a foundation for future tool application efforts. As pointed out correctly by this reviewer, this should include the question of how the local environment like presence of nucleotides and proteins may affect fluorescence lifetime, see comment below and updated Figure 2.

RESPONSE:
Additionally, we find that details about FLIM data analysis are often lost and must be systematically assessed for a rigorous comparison. Supplementary Table 3 in this study (now updated to include pilot live cell data points) serves this point. We made it more clearly in the text to highlight this point, and refer to pilot studies in cells published elsewhere in other contexts.

2. If the above understanding (this study being limited to the dyes’ characterization in parental cells which do not express the target RNA) is correct, where does the “background” come from in parental cells is an important question that the manuscript fails to discuss. Does the background come mostly from the free dye, or the dye bound to certain cellular targets (proteins or nucleic acids)? Any experiments or data to illuminate on this?

RESPONSE:
Thank you for pointing out this potential misunderstanding. The reviewer is correct that the “background” signal stems from the free probe without the bound RNA. In Riboglow-FLIM, the probe alone exhibits fluorescence stemming from the dye quenched by Cbl that leads to a distinct fluorescence lifetime signal. This signal changes when the Cbl moiety in the probe binds to the target RNA. We made this important point more clearly in the text when we introduce the Riboglow-FLIM platform (near Figure 1). We would like to point out that we performed pilot work in cells expressing the target RNA elsewhere (Sarfraz et al., 2023, Nat. Comm., Sarfraz et al., 2023 Biophys. Rep.). Part of this work was still under review while this manuscript was also in review. We have now updated these references and expanded Supplementary Table 3 to summarize all findings about Riboglow-FLIM, including live cell numbers and details about data fitting. We envision this comprehensive overview will serve as an important reference point when developing new applications of Riboglow. Added text details the importance of systematic evaluation of the free probe in different cell lines and evaluating cell health as an important foundation due to the varying intrinsic nature of different cell lines and the influence of factors such as viscosity on lifetime, as this reviewer points out correctly. For example, the fluorescence lifetime of the free probe varies when exposed to different crowding agents and additives like nucleotides and proteins (Figure 2), in line with the suggestion from this reviewer.

03-Jan-2024

Dear Dr BRASELMANN:

Manuscript ID: CB-COM-10-2023-000197.R1
TITLE: Evaluating Riboglow-FLIM probes for RNA sensing

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Reviewer 3

The authors have satisfactorily addressed the points previously raised by this reviewer.