From the journal RSC Chemical Biology Peer review history

06-May-2022

Dear Dr Manicardi:

Manuscript ID: CB-ART-04-2022-000095
TITLE: Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities:
Application to Biotin-mediated RNA-Pulldown

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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Dr Andrea Rentmeister

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

Reviewer 1

Enrico Cadoni et al. in the submitted manuscript “Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities: Application to Biotin-mediated RNA-Pulldown.” report synthesis and characterization of interstrand-crosslinking probes for sequence-specific covalent targeting of DNA and RNA. The authors show that furan-mediated crosslinking reaction is triggered by N-bromosuccinimide or light induced singlet oxygen in the presence of photosensitizers methylene blue or rhodamine B. The formed crosslink can be reversed by heating of the sample at 95 °C. The reported approach is demonstrated to work in physiological conditions, in a cell lysate, and is suitable for the sequence-specific alkylation of intracellular targets as well as pull-down assays.
Overall, this manuscript is reporting valuable probes for sequence-specific interstrand-crosslinking of DNA and RNA. Thus, I am happy to recommend this manuscript to be accepted for publishing in RSC Chemical Biology after a major revision because some additional explanations and experiments are needed:

Major points:
1. The authors tested methylene blue and rhodamine B as reactive photosensitizers. However, these fluorophores are not designed to efficiently produce singlet oxygen. For example, halogenated or selenium containing rhodamines are much more efficient chromophores for such task. It would be beneficial to show at least one example of interstrand-crosslinking using these compounds.
2. The influence of covalent bond formation for pulldown assays seems to be statistically significant but incremental. The difference between PNA-10Ac and PNA-10F seems to be relatively small. How variable this difference is in different cell lysates (biological replicates)?
3. The heating step required for reversal of covalent bond might influence stability of nucleic acid, especially RNA. Have authors estimated how much RNA is affected by heating. Are any degradation products visible? Can RNA modifications be affected by the heating step?

Minor points:
1. Please check that all abbreviations are explained before they appear in the text. For example, NBS is appearing in the abstract without being explained.
2. The text writing style is poor, and it is sometimes difficult to understand what the authors want to say. Its is important to improve it, so the readers can understand the message of the manuscript.
3. Figures 3 – 8 contain crosslinking efficiency data without error bars. Please add error bars and include information about number of performed replicates. Currently, reader cannot estimate reliability of the provided data.
4. Figures 3 – 8 contain only analysed data, and no gels were shown. Please include at least some examples of the gels. This allows the reader to estimate how well DNA or RNA is preserved after all manipulations.
5. Figure S15 and S16 show only chromatogram traces without indication which peak belongs to which compound. Please indicate this.
6. NMR spectra are without marked solvent peaks. Some 13C-NMR peaks have negative amplitude. Please mark solvent peaks and explain negative amplitudes.
7. The authors provide LC-MS data of the synthesized PNA, but do not indicate in the MS spectra which peaks belong to the target compound. Please indicate this.

Reviewer 2

In this manuscript entitled “Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities: Application to Biotin-mediated RNA-Pulldown”, Alex Manicardi and coworkers designed systematically and synthesized furan-containing PNA monomers and probes and the evaluation of their ability to form an interstrand crosslink (ICL) for the complementary nucleic acid. The effect of position of the furan unit with different types of monomers were studied. Although the designs and results are very systematic and interesting, this reviewer suggests additional experiments as listed below for publication of this manuscript in the RSC Chem Biol.

1. Thermal and thermodynamic analyses should be carried out to compare the ICL. It will be more useful if the ICL has a relationship with the thermal (thermodynamic) stability. Such thermal and thermodynamic data may provide rational reasons for the ICL, depending on positions and on the different types of the monomers.
2. Error bars with the average values are required for data shown in Figures.
3. This reviewer suggests to study effects of the nearest-neighbor pairs of the probes on the thermal stability and the ICL efficiency.
4. Is it possible to study ICL depending on the reaction time? It is important to know a sufficient incubation time for the hybridization and crosslinking.
5. Not for all the oligonucleotides designed used in this study, but for a typical one, it is interesting to study effects of cellular environmental factors such as monovalent and divalent cation concentration and molecular crowding.
6. The results and designs are systematic, it is more useful for the broad readers of the journal to conclude / propose principals for design better oligonucleotides.

see uploaded response letter

This text has been copied from the uploaded response to reviewers and does not include any figures, images or special characters:

REVIEWER REPORT(S):

Referee: 1


Comments to the Author

Enrico Cadoni et al. in the submitted manuscript “Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities: Application to Biotin-mediated RNA-Pulldown.” report synthesis and characterization of interstrand-crosslinking probes for sequence-specific covalent targeting of DNA and RNA. The authors show that furan-mediated crosslinking reaction is triggered by N-bromosuccinimide or light induced singlet oxygen in the presence of photosensitizers methylene blue or rhodamine B. The formed crosslink can be reversed by heating of the sample at 95°C. The reported approach is demonstrated to work in physiological conditions, in a cell lysate, and is suitable for the sequence-specific alkylation of intracellular targets as well as pull-down assays.
Overall, this manuscript is reporting valuable probes for sequence-specific interstrand-crosslinking of DNA and RNA. Thus, I am happy to recommend this manuscript to be accepted for publishing in RSC Chemical Biology after a major revision because some additional explanations and experiments are needed:

We thank the referee for the appreciation of our work and for their revision job, which contributed to improving the quality of the manuscript.

Major points:

1. The authors tested methylene blue and rhodamine B as reactive photosensitizers. However, these fluorophores are not designed to efficiently produce singlet oxygen. For example, halogenated or selenium containing rhodamines are much more efficient chromophores for such task. It would be beneficial to show at least one example of interstrand-crosslinking using these compounds.
As the referee correctly points out, we used methylene blue and Rhodamine B as photosensitizers to trigger the photochemical activation of the probes. Especially for what concerns rhodamine, the referee is indeed right in pointing to its low singlet oxygen quantum yield. However, as explained in the manuscript, the choice for mild/weak photosensitizers is driven by the need to control the concentration of reactive singlet oxygen, in order to ensure the selective and quantitative activation of the pro-reactive furan while avoiding oxidative degradation of the probes (i.e. biotin oxidation, as reported in our previous paper, Chem. Sci., 2020,11, 11729-11739).
Indeed, as we have previously shown, the use of rose Bengal (a halogenated-fluorescein photosensitizer with high singlet oxygen quantum yield), results in the almost quantitative oxidation of biotin to its sulfoxide. Considering the final application of our work (biotin-mediated pull-down), oxidation of the biotin tag would be deleterious as it will reduce the affinity for the streptavidin beads, hampering the efficiency of the pull-down assay (Anal. Biochem., 2017, 531, 78–82).
Experiments were performed comparing the pull-down efficiency of probes PNA-10Ac and PNA-10F, in presence of RhoB and MB. These experiments showed how the use of strong photosensitizer resulted to be detrimental to the pull-down performance of the biotin-containing probes, when compared to the use of the milder RhoB (please refer to the manuscript lines 528-34 page 17 and figure S21 in ESI).

2. The influence of covalent bond formation for pulldown assays seems to be statistically significant but incremental. The difference between PNA-10Ac and PNA-10F seems to be relatively small. How variable this difference is in different cell lysates (biological replicates)?
Following the reviewer’s suggestion, we performed additional pull-down experiments in a different cell lysate, obtained from the lysis of adenocarcinoma cancer cell line MDA-MB-231. The results obtained in these experiments showed similar trends as in SK-MEL-28 cell lysate. Also in this case, when comparing probes with identical lengths, the presence of the alkylating furan warhead showed an enhanced RNA recovery as compared to the acetylated probes. Although it might appear minor, the increase in RNA recovery under these unoptimized conditions clearly shows the benefit of introducing a (temporary) covalent modification, as the interaction between the target RNA and the pull-down probe is not lost when increasing the washing step stringency.
These new data were included in the revised version of the manuscript (Please refer to the new Figure 9c on page 18 of the manuscript, also illustrated here below).



3. The heating step required for reversal of covalent bond might influence stability of nucleic acid, especially RNA. Have authors estimated how much RNA is affected by heating. Are any degradation products visible? Can RNA modifications be affected by the heating step?

As the referee states, indeed the heating conditions could affect the sample stability and/or induce its degradation. A previous report (please refer to Nucleic Acids Research, Volume 40, Issue 18, 1 October 2012, Page e144, available at: https://doi.org/10.1093/nar/gks588) on RNA integrity shows that, upon heating at 95°C, the estimated number of lesion per 1000 bases is around 7-10, less than 1% of the RNA length. As mentioned in the submitted version of the manuscript, to confirm the integrity of the DNA target after its release, we submitted the crosslinked sample under crosslink-reversion conditions (1h at 95°C), purified the peak with HPLC, and analyzed it via MALDI-TOF. No degradation products were found. A similar experiment was now performed with RNA obtaining similar results (please refer to the new figure S20 of the revised Supporting Information, also included below).
In addition, we would like to point out that the heating step required for the release of the target from the streptavidin beads is anyway classically included in RNA pull-down protocols. We, therefore, added more emphasis on this aspect, including an additional sentence in the revised version of the manuscript (Please refer to page 14, lines 469-72: “The possibility of reverting the ICL reaction with a simple heating step proved very convenient for the final envisaged application, as heating of the streptavidin beads is a generally accepted methodology to release the probe-target complexes in common pull-down applications.”).


Figure S20. MALDI-TOF analysis of the purified RNA peak after ICL reversion upon heating the sample at 95°C for 1 hour.

Minor points:
1. Please check that all abbreviations are explained before they appear in the text. For example, NBS is appearing in the abstract without being explained.
We thank the reviewer for pointing out the issue. We, therefore, checked the manuscript for possible typos or missing acronym definitions and corrected/defined them in the revised version of the manuscript.

2. The text writing style is poor, and it is sometimes difficult to understand what the authors want to say. Its is important to improve it, so the readers can understand the message of the manuscript.
Following the referee’s suggestion, we have asked a native English speaker to check and revise the manuscript where necessary. The text was revised accordingly.

3. Figures 3 – 8 contain crosslinking efficiency data without error bars. Please add error bars and include information about number of performed replicates. Currently, reader cannot estimate reliability of the provided data.
In the initial phase, when screening a large series of crosslink conditions, all experiments were performed as single replicates, as we aimed at generating a general qualitative overview of the alkylation trends of the different probes under all different conditions. Triplicate experiments were then performed in the finally chosen setup.
Accordingly, error bars are added to figure 8 and the captions of each figure adapted to indicate the number of replicates.

4. Figures 3 – 8 contain only analysed data, and no gels were shown. Please include at least some examples of the gels. This allows the reader to estimate how well DNA or RNA is preserved after all manipulations.

5. Figure S15 and S16 show only chromatogram traces without indication which peak belongs to which compound. Please indicate this.
We thank the reviewer for pointing this issue out. We updated the figure with the indications needed. Please refer to the new figures S18 and S19 of the revised ESI.

6. NMR spectra are without marked solvent peaks. Some 13C-NMR peaks have negative amplitude. Please mark solvent peaks and explain negative amplitudes.

The chemical shift indication of the solvent residual peak was removed from crowded spectra, after signal calibration, to avoid misinterpretation of other signals that share the same chemical shift (e.g. Molecule 24 in Figure S43). The residual solvents’ peak indications were additionally removed from less crowded spectra in order to homogenize data presentation.
Following the reviewer’s comment, we
 
Referee: 2

Comments to the Author
In this manuscript entitled “Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities: Application to Biotin-mediated RNA-Pulldown”, Alex Manicardi and coworkers designed systematically and synthesized furan-containing PNA monomers and probes and the evaluation of their ability to form an interstrand crosslink (ICL) for the complementary nucleic acid. The effect of position of the furan unit with different types of monomers were studied. Although the designs and results are very systematic and interesting, this reviewer suggests additional experiments as listed below for publication of this manuscript in the RSC Chem Biol.

1. Thermal and thermodynamic analyses should be carried out to compare the ICL. It will be more useful if the ICL has a relationship with the thermal (thermodynamic) stability. Such thermal and thermodynamic data may provide rational reasons for the ICL, depending on positions and on the different types of the monomers.
We would like to point out that the crosslinking ability of the probes does not depend on the thermal (or thermodynamic) stability of the resulting duplexes, as long as the NA:PNA complexes are formed under experimental conditions (25°C, 100 mM NaCl, 10 mM Phosphate, pH 7.4). It is rather the position of the reacting nucleophilic residues at the moment of furan activation that determines the crosslink efficiency. With that in mind, probes were designed so that the complementary regions granted sufficient stability to the complexes to be formed, even in presence of mismatched residues. This was confirmed by melting experiments, reported here below (data not shown in the manuscript).
We additionally added a sentence in the manuscript, indicating that the formation of a complex even in presence of doubly-mismatched sequences, was taken into account. Please refer to page 8 lines 233-35 of the revised version of the manuscript: “DNA:PNA complex formation was confirmed on representative samples (containing one or two mismatches) via UV-melting experiments (data not shown).”
Representative melting curves of samples containing PNA:DNA duplexes with two (top) or one mismatch (bottom), performed at 5 μM probe concentration in PBS (100 mM NaCl, 10 mM Phosphate, pH 7.4). The melting experiments were performed in duplicate (blue curve and red curve).


2. Error bars with the average values are required for data shown in Figures.
As discussed in the answer to comment 3 of referee 1, in the initial screening of crosslink conditions only single experiments were performed. This was done to quickly understand the behavior of the probes and to obtain an idea of the general alkylation trend. Therefore, not all the experiments were performed in replicate.
Even if not explicitly asked by the reviewer, we repeated in triplicate the alkylation experiment reported in figure 8, and the error bars were added accordingly. Finally, the numerosity of each experiment set was reported in the caption of the corresponding figure of the manuscript.
3. This reviewer suggests to study effects of the nearest-neighbor pairs of the probes on the thermal stability and the ICL efficiency.

We agree with the referee that studying the effect of the nearest neighbor pair on the ICL efficiency and thermal stability could be interesting and is indeed described in some of our previous reports (cfr. J. Am. Chem. Soc.2011, 133, 4, 796–807 and Nucleic Acids Research, Volume 37, Issue 5, 1 April 2009, Pages 1555–1565). However, for this specific case, this is a considerable amount of work, which is outside the scope of the present paper. The total number of experiments and amount of synthetic work to be performed to completely evaluate this factor would require an extensive effort. Only for the internal modifications, 64 PNA probes would need to be evaluated for each modified monomer (in the present work, we tested 4 permutations) and a total of 256 DNA targets (in the present work we included 16), accounting for 16384 combinations (therefore 16320 additional experiments). Considering that in the final application of the work the terminally-modified PNAs (that showed a broad reactivity and higher ICL%, as demonstrated by the C-Scan experiments) are exploited, we believe that the results would not be useful enough for the reader to justify such an extensive effort.

4. Is it possible to study ICL depending on the reaction time? It is important to know a sufficient incubation time for the hybridization and crosslinking.
We understand the point of the reviewer, but unfortunately, it is rather difficult to evaluate.
In a previous work on DNA-templated PNA ligation (Chem. Sci., 2020,11, 11729-11739), we tried to disconnect the two processes of furan activation and subsequent ligation by quenching the reaction with methylhydrazine following a previously developed protocol (Chem. - A Eur. J., 2016, 22, 8457–8461). This however resulted in very poor gel and HPLC quality not allowing to obtain specific information on the yield and kinetics of the oxidation only. Concerning the hybridization time, this is also a difficult point to evaluate as for these short sequences the process is rather fast even in presence of sequence mismatches (Biochemistry 1997, 36, 16, 5072–5077).

5. Not for all the oligonucleotides designed used in this study, but for a typical one, it is interesting to study effects of cellular environmental factors such as monovalent and divalent cation concentration and molecular crowding.
Following the reviewer’s suggestion, we performed an experiment in which high concentration of various cations were employed (200 mM). For simulating high molecular crowding conditions, we used PEG6000 at a final concentration of 30% at a physiological potassium concentration of 150 mM in presence of 10 mM sodium and 0.5 mM magnesium. As illustrated in the figure included below, the outcome of the ICL reaction did not significantly change when varying the composition of the reaction mixture. We therefore included a sentence in the manuscript (refer to page 14, line 455-59 of the revised version: “Crosslinking was additionally studied in presence of a high concentration of various ions (Ca2+, Mg2+, K+), as well as high molecular crowding conditions to simulate the physiological concentration of species in living cells, using PEG6000 (30% w/v). In none of these cases, the outcome of the reaction appeared to be influenced (please refer to ESI, Figure S2)”) and included the figure below in the supporting information of the revised version of the paper:

Figure S2. ICL reaction performed in presence of DNA-2-A and PNA-5, using MB as photosensitizer at a final concentration of 2 μM, varying solution composition. Experiments performed at 5 μM final strand concentration, in PBS buffer pH 7.4 supplemented with 400 mM Ca2+ (A), 200 mM Mg2+ (B), 200 mM K+ (C) or in a 30% PEG6000 solution supplemented with 150 mM K+, 10 mM Na+ and 0.5 mM Mg2+.

6. The results and designs are systematic, it is more useful for the broad readers of the journal to conclude / propose principals for design better oligonucleotides.
Following the referee’s suggestion, we inserted an extra paragraph to discuss the preliminary ICL results. Please, refer to page 14 of the revised manuscript, lines 430-39:
Summarizing the results obtained in the preliminary ICL experiments, to maximize the ICL yield, alkylating probes based on terminal incorporation of universal monomers M1 and M4 should be used. These probes showed high reactivity and good tolerance to different activation protocols. The possibility to use these modifications to target all reactive nucleobases placed from N-5 to N-1 position, with a preference for cytosine (where ICL % was higher than 90%), allows great flexibility in the design of pull-down probes. Higher sequence selectivity was obtained when the reactive furan was placed in a central position of the PNA probe, especially using M2 and M3. Unfortunately, the low tolerance of these modifications to singlet oxygen discourages their use in applications where NBS activation is not allowed. Finally, the incorporation of M1 in a central position showed intermediate properties.

18-Jul-2022

Dear Dr Manicardi:

Manuscript ID: CB-ART-04-2022-000095.R1
TITLE: Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities:
Application to Biotin-mediated RNA-Pulldown

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Reviewer 2

This manuscript have been improved according to the reviewers' comments. This reviewer is glad to support this manuscript for publication in RSC Chem Biol.