From the journal RSC Chemical Biology Peer review history

21-Dec-2023

Dear Dr Helm:

Manuscript ID: CB-ART-11-2023-000221
TITLE: Reversible oxidative dimerization of 4-thiouridines in tRNA isolates

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.

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Yours sincerely,
Claudia Höbartner
Associate Editor, RSC Chemical Biology

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Reviewer 1

Bessler et al. have studied nucleosides from E. coli tRNA isolates by LC-MS, and report the observation of disulfide-bridged dimers of 4-thiouridine (s4U). By careful characterization of (M+Na)+ and (M+K)+ ions at m/z 541 and 557 using neutral loss scans (triple quadrupole MS), stable isotope labeling (13C, 15N, 34S), high resolution MS (Q-TOF), disulfide bond reduction (DTT) and thiol oxidation (formation of disulfide-bridged tRNA), they were able to show that the disulfide-bridged dimers of s4U formed after tRNA isolation, i.e., during sample workup and/or storage in the presence of ambient oxygen.

This study is of great importance for the use of thiouridine as a metabolic label, for thiouridine-to-cytidine conversion sequencing, and for the study of the underexplored thiol chemistry of RNA in general. The manuscript is clear and beautifully illustrated, and I recommend publication as is.

Reviewer 2

Here the authors identified a potential novel nucleoside using neutral loss scans within E. coli tRNA isolates and aimed to elucidate the structure. The two chromatographic peaks of interest exhibited the same ions, fragmentation, and chemical formula confirmed through stable isotope labeling. They identified the modification as an s4U dimer connected through a disulfide linkage which was confirmed through DTT treatment and I2-KI treatment of synthetic s4U nucleosides with comparison of LC-MS/MS data. The authors then determined if the s4U dimer was a native RNA modification or if it was an artifact of sample preparation. The data from the mixed 14N and 15N analysis exhibits an s4U dimer with a peak representative of a 14N-s4U linked to a 15N-s4U; thus, confirming that the s4U dimer is an artifact of sample preparation and not an RNA modification generated in vivo. This is a well-written manuscript; however, it would benefit from further clarification, see below.
1. The sentence “The data set of potentially new ribonucleoside structures included four candidates, of which two each showed M+1…” is unclear and would benefit from rephrasing in highlighting that two chromatographic peaks each have the same two ions, 541 and 557.
2. The authors reference the NLS dataset and identify a small peak for m/z 519 indicative of the M+H for the ions of interest. High-resolution MS data shows an abundant 519 peak with the M+Na and M+K ions at a lower abundance. It is unclear why there is a difference in the m/z 519 ion abundance between the two methods. The addition of a line explaining this difference would benefit the manuscript.
3. Like the above comment, the authors chose to elucidate the structure of the M+Na ion. A line explaining why the M+H ion was not chosen for structural elucidation would improve clarity for the reader.
4. Lastly, the authors reference Occam’s razor in that the dimer is most likely an in vitro artifact. The data represented in Figure 4 shows that the dimer is formed in vitro through the formation of a 14N and 15N s4U dimer. Therefore, this conclusion using Occam’s razor decreases the impact of the conclusions drawn from the data shown in Figure 4. I recommend the removal of this point.

Responses to Reviewers‘ Comments
We thank the reviewers for their fair verdict and competent comments. We have addressed the minor issues as detailed below.

Referee: 1
Comments to the Author
Bessler et al. have studied nucleosides from E. coli tRNA isolates by LC-MS, and report the observation of disulfide-bridged dimers of 4-thiouridine (s4U). By careful characterization of (M+Na)+ and (M+K)+ ions at m/z 541 and 557 using neutral loss scans (triple quadrupole MS), stable isotope labeling (13C, 15N, 34S), high resolution MS (Q-TOF), disulfide bond reduction (DTT) and thiol oxidation (formation of disulfide-bridged tRNA), they were able to show that the disulfide-bridged dimers of s4U formed after tRNA isolation, i.e., during sample workup and/or storage in the presence of ambient oxygen.

This study is of great importance for the use of thiouridine as a metabolic label, for thiouridine-to-cytidine conversion sequencing, and for the study of the underexplored thiol chemistry of RNA in general. The manuscript is clear and beautifully illustrated, and I recommend publication as is.

Response 1
Thank you very much.


Referee: 2
Comments to the Author
Here the authors identified a potential novel nucleoside using neutral loss scans within E. coli tRNA isolates and aimed to elucidate the structure. The two chromatographic peaks of interest exhibited the same ions, fragmentation, and chemical formula confirmed through stable isotope labeling. They identified the modification as an s4U dimer connected through a disulfide linkage which was confirmed through DTT treatment and I2-KI treatment of synthetic s4U nucleosides with comparison of LC-MS/MS data. The authors then determined if the s4U dimer was a native RNA modification or if it was an artifact of sample preparation. The data from the mixed 14N and 15N analysis exhibits an s4U dimer with a peak representative of a 14N-s4U linked to a 15N-s4U; thus, confirming that the s4U dimer is an artifact of sample preparation and not an RNA modification generated in vivo. This is a well-written manuscript; however, it would benefit from further clarification, see below.

2.1
The sentence “The data set of potentially new ribonucleoside structures included four candidates, of which two each showed M+1…” is unclear and would benefit from rephrasing in highlighting that two chromatographic peaks each have the same two ions, 541 and 557.

Response 2.1:
In order to make the information clearer, we rephrased the respective section of the text as follows: “At the onset of the present study, within the data set of potentially new ribonucleoside structures, we noticed an early eluting peak at 14.6 min and a later eluting peak at 25.7 min in our reversed-phase chromatography which both were characterised by the simultaneous detection of the same two ions with a mass-to-charge ratio of 541 and 557, respectively (Figure 1a+d).”

2.2
The authors reference the NLS dataset and identify a small peak for m/z 519 indicative of the M+H for the ions of interest. High-resolution MS data shows an abundant 519 peak with the M+Na and M+K ions at a lower abundance. It is unclear why there is a difference in the m/z 519 ion abundance between the two methods. The addition of a line explaining this difference would benefit the manuscript.

Response 2.2:
The difference in the m/z 519 ion abundance between the LC-QQQ and the LC-Q-ToF was surprising for us to observe, but unfortunately, we were not really able to determine the crucial factors behind this behaviour. However, we assume that this is most likely a system specific phenomenon but it might also be traced to different batches of mobile phase solvents. Interestingly, we found a manuscript (Site-Specific Profiling of 4-Thiouridine Across Transfer RNA Genes in Escherichia coli | ACS Omega; Figure 3c) in which s4U was analysed by LC-MS (in that case HRMS with a Q Exactive mass spectrometer) that was indicative of a predominant occurrence of the sodium adduct of s4U instead of its protonated species. Considering this, we speculate that detection of s4U is vulnerable to even smallest traces of sodium in any LC-MS system or solvent which assumingly might also apply for the dimeric species.
We inserted the following sentence at the respective position in the text, in order to make these thoughts more transparent for the readers:

“Interestingly, there was a very pronounced difference in the distribution of protonated versus adduct species when comparing analyses from the HRMS instrument (Figure 2b) and the QQQ instrument (Supplemental Figure S2) which is most likely a system specific phenomenon but might also be traced to smallest changes in different batches of the mobile phases.”

2.3
Like the above comment, the authors chose to elucidate the structure of the M+Na ion. A line explaining why the M+H ion was not chosen for structural elucidation would improve clarity for the reader.

Response 2.3:
To improve clarity, we added an explanation why the M+Na ion was chosen for structural elucidation:
“Since QQQ measurements were used for further MS analyses and both candidates were shown to be adducts of the same compound with the sodium adduct being the most abundant species during QQQ analysis, further characterisation experiments are representatively shown for this candidate.”

2.4
Lastly, the authors reference Occam’s razor in that the dimer is most likely an in vitro artifact. The data represented in Figure 4 shows that the dimer is formed in vitro through the formation of a 14N and 15N s4U dimer. Therefore, this conclusion using Occam’s razor decreases the impact of the conclusions drawn from the data shown in Figure 4. I recommend the removal of this point.

Response 2.4:
We now removed the mention of Occam’s razor and changed the respective part of the text as follows:
“Given that in vivo conditions are typically reductive, and that we have recapitulated reductive cleavage of the dimer by thiols,44,47 we conclude that the dimerization took place in vitro, presumably during work-up and storage, with the oxidative dimerization likely caused by ambient oxygen.”

31-Jan-2024

Dear Dr Helm:

Manuscript ID: CB-ART-11-2023-000221.R1
TITLE: Reversible oxidative dimerization of 4-thiouridines in tRNA isolates

Thank you for submitting your revised manuscript to RSC Chemical Biology. I am pleased to accept your manuscript for publication in its current form.

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