From the journal RSC Chemical Biology Peer review history

19-Feb-2022

Dear Mr Argueta-Gonzalez:

Manuscript ID: CB-ART-01-2022-000020
TITLE: Stimuli-Responsive Assembly of Bilingual Peptide Nucleic Acids

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.

I have carefully evaluated your manuscript and the reviewers’ reports, and the reports indicate that major revisions are necessary. Three reviewers raised concerns about the interpretation of your data and advise that more experimental evidence is needed to support the central claims of the work.

I invite you to submit a revised manuscript which addresses all of the reviewers’ comments. Further peer review of your revised manuscript may be needed. 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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Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg

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Reviewer 1

The authors previously reported the formation of nanostructures of peptide nucleic acid (PNA) modified with side chain leading to an amphiphilic sequence. The presence of a hydrophobic part and hydrophilic part led to the formation of nanostructures in water. It was demonstrated that the addition of a complementary DNA sequence induced the disassembly. In the current manuscript the authors studied the reverse process. A DNA sequence partially hybridized with the modified PNA was displaced with a fully complementary DNA to release the PNA allowing it to be arranged into nanostructures.
First the displacement was study with a standard PNA (PNA-C1-FAM). It has been demonstrated that the displacement of the PNA-C1-FAM masked with a DNA sequence was more efficient with a DNA releasing sequence than with a RNA one while when a RNA masked sequence was used both releasing sequences displayed the same efficiency. However, a slower of kinetic was observed in this latter case. Surprisingly the displacement of the masking sequence with a mismatch was less efficient than the full match sequences.
It is known that mismatches are more destabilizing in the sequence than at the extremity so it is not surprising that the effect on the stabilization of duplex is moderate. It will be more interesting to study a mismatch in the middle of the sequence to see if the dehybridization kinetic are faster.

Then the displacement studied with the modified PNA exhibiting hydrophilic and hydrophobic blocks. The displacement of PNA-A1-FAM masked with a DNA sequence was again found more efficient with a releasing DAN sequence than with a RNA one.
Disassembly and reassembly was monitored by DLS but it was shown that only large size particles were formed due the participation of the DNA sequence whatever the structures expected. Thus this method was not appropriated to study the rearrangements obtained in this case.

The CD study is very confusing. Why did the names of the sequences change? What are PNA-A, RNA, DNA and RS-D-0? It is difficult to understand what has been observed. Please clarify this study and better explained the conclusion.

For the TEM study, why the PAN-A1-FAM alone was not studied at 100 µM like for the two other conditions? Since the conditions of concentration for Fig A and Fig 7C are different it is difficult to compare. So we cannot conclude if the differences observed by TEM are due to the differences of concentration or to the presence of DNA duplex.

According to the data presented in this manuscript I'm not convinced that the system is reversible by the addition of the releasing DNA to yield the assembled PNA-A1-FAM alone. It seems that by addition of the releasing DNA, the system resulted in a different structure involving both PNA-A1-FAM and the duplex formed in situ as shown by DLS and TEM.
See the guide for authors for the format of references.

In summary the current manuscript reported interesting results but some points need to be clarified.

Reviewer 2

The manuscript describes an extension of the authors' previous work on bilingual PNA (ref 16), whereby the PNA molecule was modified to have distinct hydrophobic and hydrophilic domains (via the installation of amino acid side-chains at the gamma positions) so that it can self-assemble into a micelle-like structure. The structure was previously shown to disassemble upon hybridization with its complementary nucleic acid sequence. This work went on further to demonstrate that the hybridized bilingual PNA can be displaced from its complementary DNA/RNA strand via a toehold-mediated strand displacement with another DNA/RNA strand. Thus, the aggregation states of the PNA could, in principle, be controlled reversibly by the addition of the appropriate DNA/RNA sequences. Fluorescence experiments showed that the strand displacement had indeed occurred, as shown by the restoration of the fluorescence of the PNA probe. There are some subtle kinetics differences between DNA and RNA masking strands and releasing strands. The most efficient pairs seem to be the DNA both as masking strands and releasing strands. The specificity of the strand displacement process was also demonstrated. However, the fluorescence assay does not give critical information about the aggregation states of the displaced PNA probe as essential the same results were obtained for both normal (PNA-A1-FAM) and bilingual (PNA-C1-FAM) PNA probes. Other attempts to show the aggregation states by DLS, CD, and TEM did not give an unequivocal result. The DLS showed that the disassembled PNA was as larges as the reassembled PNA probes, and both were even larger than the PNA probe before the disassembling process. TEM experiments were performed under concentrations below CMC, so it was not clear whether this truly represents the real situation. CD experiments are rather confusing as the PNA and DNA/RNA used are different from the ones being used in other experiments. Also, the "PNA:RNA + DNA" signal changes only slightly when compared to the "PNA:RNA" signal. At equilibrium, the system should consist of DNA:RNA + PNA, and thus the signal should be more or less the same as "RNA:DNA+PNA". This seems to suggest that the strand displacement occurred only partially. Most importantly, the CD experiment still does not provide direct evidence of the reassembling of the displaced PNA probe. Overall, although there is nothing wrong with the working principle and this would make a great story, the evidence for the reassembling of the bilingual PNA following the strand displacement process is still missing. A more solid proof should be required before the manuscript can be accepted.


Other comments:
- The mismatched sequences (MS-D-m1 and RS-D-m1 in Table 1) were introduced with the hope to facilitate the kinetics of the displacement process by destabilizing the PNA-DNA duplex, but it was rather counterintuitive to place the mismatch base at the 3'-end of the DNA strand instead of at internal positions. It is thus no surprise that the stability of the mismatched PNA-DNA was not much different from the complementary PNA-DNA duplexes. What is the rationale behind this choice of mismatch placement?

- p6, last line: 317 nm -> 317 nM.
- Fig. 4 caption (D) PNA-C1-FAM:MSD was missing from the caption, only PNA-A1-FAM:MSD was mentioned. The caption also referred to a scrambled DNA sequence RS-D-sc, but its sequence was missing. This should be added here or in Table 1.
- Fig 6 and the accompanying discussion: This is difficult to follow as the PNA/DNA/RNA involved are different from the sequences used in Table 1. Why didn't the authors use the same set of PNA/DNA/RNA? Also, the RS-D-0 in the caption is not defined anywhere else in the manuscript.
- The structure of FAM in Fig. S7 was wrong. The oxygen atom in the xanthene ring was missing.
- Table S2. The expected mass of PNA-A1-FAM should be shown with the same decimal place as other sequences.
- Table S2. Surprisingly, the mass difference of the PNA-C1-FAM and PNA-A1-FAM was only 40 Da. I expect the 2xala and 2xlys side chains would add up more mass.
Please check the figure carefully.
- Fig. S10 has not been mentioned in the main manuscript.

Reviewer 3

The manuscript describes the use of toehold-mediated strand displacement to induce aggregation of PNAs functionalized with amino acid side chains. The authors refer to these compounds as bilingual PNAs because of the presence of both amino acid side chains and nucleobases. In a 2019 JACS paper, they described the self-assembling properties of a PNA having two alanine and two lysine side chains functionalized at the gamma carbon, interspersed along the PNA sequence. The current manuscript extends this work by first masking the PNA via hybridization to a complementary DNA or RNA, which is intended to prevent aggregation of the PNA, but then releasing the mask via toehold-mediated strand exchange to induce aggregation. This is a logical extension but there are some surprising results along the way. Overall, readers should find the manuscript interesting although there are a lot of loose ends and I’m not convinced that the authors have interpreted their DLS results correctly. The authors should consider the following points to improve the manuscript.

1. It appears that a single amino acid sequence (AAKK) was used in both the 2019 JACS paper and the current manuscript so it is unclear to what extent the sequence of amino acid side chains is important for the observed phenomena. Without multiple sequences, should the PNA actually be called bilingual?

2. I’m fully supportive of giving Rosalind Franklin the credit she deserves, but it doesn’t seem appropriate to do so in the context of base pairing. Erwin Chargaff and Jerry Donohue had much more to do with Watson and Crick getting G-C and A-T to pair properly whereas Franklin’s data was essential for deducing the double helical structure of DNA. I would rather see “Watson-Crick-Franklin double helix” than “Watson-Crick-Franklin base pairs”, but I’m probably swimming upstream here.

3. In table 1, readers might confuse the C-terminus (designated “C”) as a cytosine. Perhaps underline or italicize C and N at the termini?

4. In Figure 2, it would be helpful to include the PNA concentration in the caption.

5. Why use a 10 nucleotide overhang? My understanding of the literature is that the displacement rate maximizes at toehold length of around 5 nucleotides.

6. Why place the mismatch at the 3’-end of the masking sequence when an internal mismatch is more likely to cause destabilization?

7. Bizarre result #1: the authors find that the mismatch leads to higher PNA-DNA melting temperature. Good for the authors to include this and point it out to the readers, but it does leave one wondering why the Tm increased for a T-T mismatch.

8. I realize that RNA-DNA typically has lower Tm than DNA-DNA but the large difference in % Displacement for RNA vs DNA (Figure 3B) is surprising to me. Is this bad intuition on my part or is there some other explanation, such as the experiment being done close to the Tm for RNA-DNA, magnifying the affinity difference to a much larger degree than if the experiment were done well below both Tms?

9. At the top of page 9, the authors state that differences in concentration, stoichiometry and buffer conditions could explain why they observe an opposite trend for RNA releasing sequences compared to predictive models. I can see how experimental differences might change the magnitude of an effect, but reversing it is harder to understand.

10. Bizarre result #2: In Figure 4C, why is there no effect on % Displacement of going from 1.0 to 1.5 equivalents of RS-R but then the displacement increased from 1.5-2.0 equivalents?

11. In Figure 6, PNA-A1-FAM is shown at 100 uM whereas the other samples are shown at 10 uM. It seems that PNA-A1-FAM alone should also be shown at 10 uM.

12. I don’t know what to make of the DLS experiments, which seem to contradict the central hypothesis of the work, namely that adding a masking strand would prevent aggregation when what is observed is a 10-fold increase in particle size. Moreover, addition of the displacing strand has no effect on the size. Rather than conclude that their approach doesn’t work, the authors instead decide that DLS isn’t appropriate for measuring the effects and resort to TEM. However, TEM only reflects what was deposited on the grid and in fact the grid and uranyl acetate stain could be influencing the equilibria. Also, the concentrations of the DLS and TEM experiments were quite different. I guess I’m left with doubts about whether this elegant design actually works.

RESPONSES TO REVIEWERS’ COMMENTS

REVIEWER 1

Reviewer Comment: It will be more interesting to study a mismatch in the middle of the sequence to see if the dehybridization kinetic are faster.

Response: We agree with the reviewer that introducing a mismatch in the middle of the sequence could also be interesting, but we were concerned about impact on the hybridization stability and assembly properties. We have added a brief rationale to the manuscript for our choice of mismatch location:

We chose to locate the mismatch at a terminal site rather than an internal site for two reasons. First, internal mismatches in PNA can have a significant impact on duplex stability and we recognized that this would likely prevent efficient hybridization at room temperature. Second, the primary role of the masking sequence is to shield the hydrophobic block of the PNA sequence, and thus we sought to locate the mismatch as far away as possible from this region of the duplex.

Reviewer Comment: Disassembly and reassembly was monitored by DLS but it was shown that only large size particles were formed due the participation of the DNA sequence whatever the structures expected. Thus, this method was not appropriated to study the rearrangements obtained in this case.

Response: We agree that DLS did not provide conclusive results for the disassembled and reassembled samples. This was not entirely unexpected, given our previous observation of large non-specific aggregates upon addition of DNA or RNA to PNA samples, coupled with the fact that large assemblies can dominate the DLS signal, prohibiting detection of smaller assemblies. To provide greater clarity, we have moved these DLS results to the SI and revised the manuscript to better explain that the TEM results are what provide evidence for disassembly and reassembly.

Reviewer Comment: The CD study is very confusing. Why did the names of the sequences change? What are PNA-A, RNA, DNA and RS-D-0? It is difficult to understand what has been observed. Please clarify this study and better explained the conclusion.

Response: We apologize for this confusion. The CD study was initially performed using our PNA sequence having a DMN fluorophore. We have repeated these experiments with the same sequences used in the toehold-mediated displacement and updated Figure 6 in the manuscript.

Reviewer Comment: For the TEM study, why the PAN-A1-FAM alone was not studied at 100 µM like for the two other conditions? Since the conditions of concentration for Fig A and Fig 7C are different it is difficult to compare. So we cannot conclude if the differences observed by TEM are due to the differences of concentration or to the presence of DNA duplex.

Response: We initially used 100 µM for our samples having PNA alone, but later changed to 10 µM upon observing non-specific aggregation with addition of DNA and RNA. We agree with the Reviewer that the same concentration should be used for all samples. We have collected TEM images for PNA-A1-FAM at 10 µM and added these to the manuscript. All images at 100 µM are now available in the SI.

Reviewer Comment: According to the data presented in this manuscript I'm not convinced that the system is reversible by the addition of the releasing DNA to yield the assembled PNA-A1-FAM alone. It seems that by addition of the releasing DNA, the system resulted in a different structure involving both PNA-A1-FAM and the duplex formed in situ as shown by DLS and TEM.
See the guide for authors for the format of references.

Response: While the DLS data are inconclusive, we feel that the TEM data do demonstrate disassembly and stimuli-responsive reassembly. Specifically, while we observe large aggregates upon addition of DNA and RNA (similar to in our previous work), we also see the disappearance of the ordered PNA assemblies upon addition of masking strand, followed by reappearance of these assemblies upon addition of releasing strand. We have elaborated upon our discussion in the manuscript to highlight this point:

While we do still observe some non-specific aggregates, we were excited to also observe a large number of ordered assemblies having an average size of 16.8 ± 5.7 nm (Fig. 7C). We do note that the structures formed upon reassembly are slightly smaller than those formed in the initial PNA assembly. This may be due to the presence of DNA in the solutions or as a result of a different assembly mechanism when the PNA is being released from a DNA complement over time. However, we were excited to observe that our bilingual PNA is able to undergo stimuli-responsive assembly as anticipated.

REVIEWER 2

Reviewer Comment: However, the fluorescence assay does not give critical information about the aggregation states of the displaced PNA probe as essential the same results were obtained for both normal (PNA-A1-FAM) and bilingual (PNA-C1-FAM) PNA probes. Other attempts to show the aggregation states by DLS, CD, and TEM did not give an unequivocal result. The DLS showed that the disassembled PNA was as large as the reassembled PNA probes, and both were even larger than the PNA probe before the disassembling process. TEM experiments were performed under concentrations below CMC, so it was not clear whether this truly represents the real situation.

Response: The fluorescence assay was used to determine the % hybridized and was not intended to determine the aggregation state. We agree that the DLS data for the disassembled and reassembled samples were not conclusive, and thus were moved to the SI. However, we feel that the TEM data do demonstrate disassembly and stimuli-responsive reassembly. Specifically, while we observe large aggregates upon addition of DNA and RNA (similar to in our previous work), we also see the disappearance of the ordered PNA assemblies upon addition of masking strand, followed by reappearance of these assemblies upon addition of releasing strand. We also highlight that these studies were performed at 100 µM, which is above the CMC of 317 nM. We have elaborated upon our discussion in the manuscript to highlight this point:

While we do still observe some non-specific aggregates, we were excited to also observe a large number of ordered assemblies having an average size of 16.8 ± 5.7 nm (Fig. 7C). We do note that the structures formed upon reassembly are slightly smaller than those formed in the initial PNA assembly. This may be due to the presence of DNA in the solutions or as a result of a different assembly mechanism when the PNA is being released from a DNA complement over time. However, we were excited to observe that our bilingual PNA is able to undergo stimuli-responsive assembly as anticipated.

Reviewer Comment: CD experiments are rather confusing as the PNA and DNA/RNA used are different from the ones being used in other experiments.

Response: We apologize for this confusion. The CD study was initially performed using our PNA sequence having a DMN fluorophore. We have repeated these experiments with the same sequences used in the toehold-mediated displacement and updated Figure 6 in the manuscript.

Reviewer Comment: Also, the "PNA:RNA + DNA" signal changes only slightly when compared to the "PNA:RNA" signal. At equilibrium, the system should consist of DNA:RNA + PNA, and thus the signal should be more or less the same as "RNA:DNA+PNA". This seems to suggest that the strand displacement occurred only partially.

Response: We agree that the CD of the reassembled system should show the combination of DNA:RNA + PNA and we are pleased that this is what we observe when repeating these studies with our current sequences. These data are in revised Fig. 6 in the manuscript. We have also added a comment to this effect in the manuscript:

After introduction of the RS-D strand to the PNA-A1-FAM:MS-D duplex, we observed a shift in signal, indicating that the PNA amphiphile was released. Additionally, the signal observed after addition of the releasing sequence is very similar to a combination of signals from PNA alone and the MS-D:RS-D duplex, providing additional evidence for successful toehold-mediated displacement.

Reviewer Comment: Most importantly, the CD experiment still does not provide direct evidence of the reassembling of the displaced PNA probe.

Response: The CD experiment was intended to show that a change in hybridization state occurs upon addition of masking strand and releasing strand. Reassembly of the displaced PNA probe is established using TEM, as this is the best suited method for directly observing the expected PNA assemblies.

Reviewer Comment: Overall, although there is nothing wrong with the working principle and this would make a great story, the evidence for the reassembling of the bilingual PNA following the strand displacement process is still missing. A more solid proof should be required before the manuscript can be accepted.

Response: We recognize that our explanation of the DLS and TEM data in the initial manuscript may have led to this concern. We have improved the clarity of this discussion and feel that the disappearance and reappearance of the PNA assemblies (not to be confused with the non-specific aggregates) in the TEM images provide strong support that the system is functioning as anticipated.

Reviewer Comment: Mismatched sequences (MS-D-m1 and RS-D-m1 in Table 1) were introduced with the hope to facilitate the kinetics of the displacement process by destabilizing the PNA-DNA duplex, but it was rather counterintuitive to place the mismatch base at the 3'-end of the DNA strand instead of at internal positions. It is thus no surprise that the stability of the mismatched PNA-DNA was not much different from the complementary PNA-DNA duplexes. What is the rationale behind this choice of mismatch placement?

Response: We agree with the reviewer that introducing a mismatch at the terminus might be expected to have attenuated impact, but we were concerned about the impact on hybridization stability and assembly properties if we utilized an internal mismatch. We have added a brief rationale to the manuscript for our choice of mismatch location:

We chose to locate the mismatch at a terminal site rather than an internal site for two reasons. First, internal mismatches in PNA can have a significant impact on duplex stability and we recognized that this would likely prevent efficient hybridization at room temperature. Second, the primary role of the masking sequence is to shield the hydrophobic block of the PNA sequence, and thus we sought to locate the mismatch as far away as possible from this region of the duplex.

Reviewer comment:

- p6, last line: 317 nm -> 317 nM.
- Fig. 4 caption (D) PNA-C1-FAM:MSD was missing from the caption, only PNA-A1-FAM:MSD was mentioned. The caption also referred to a scrambled DNA sequence RS-D-sc, but its sequence was missing. This should be added here or in Table 1.
- Fig 6 and the accompanying discussion: This is difficult to follow as the PNA/DNA/RNA involved are different from the sequences used in Table 1. Why didn't the authors use the same set of PNA/DNA/RNA? Also, the RS-D-0 in the caption is not defined anywhere else in the manuscript.
- The structure of FAM in Fig. S7 was wrong. The oxygen atom in the xanthene ring was missing.
- Table S2. The expected mass of PNA-A1-FAM should be shown with the same decimal place as other sequences.
- Table S2. Surprisingly, the mass difference of the PNA-C1-FAM and PNA-A1-FAM was only 40 Da. I expect the 2xala and 2xlys side chains would add up more mass.
Please check the figure carefully.
- Fig. S10 has not been mentioned in the main manuscript.

Response: We thank the reviewer for their attention to detail. These errors have been corrected.
We also appreciate the reviewer for suggesting a review of the PNA oligomer mass. We found that the mass was correct and that the difference in 40 Da comes from the lysine residue and amino acid backbone on PNA-C1-FAM, introducing 145 Da, while the PNA-A1-FAM has 2 lysine residues only (72 Da each) and 2 alanine residues (14 Da each) only.

REVIEWER 3

Reviewer Comment: It appears that a single amino acid sequence (AAKK) was used in both the 2019 JACS paper and the current manuscript so it is unclear to what extent the sequence of amino acid side chains is important for the observed phenomena. Without multiple sequences, should the PNA actually be called bilingual?

Response: We appreciate this comment, but highlight that the “bilingual” nature is due to encoding both amino acid and nucleobase languages, not from having multiple different sequences. In our initial paper, we did also explore a sequence having the amino acids grouped at the PNA termini and observed that this did not result in the same assembly properties. We also have work underway to explore bilingual PNAs having a variety of different nucleotide and amino acid sequences.

Reviewer Comment: In table 1, readers might confuse the C-terminus (designated “C”) as a cytosine. Perhaps underline or italicize C and N at the termini?

Response: This is a good point. We have updated the Table to annotate the C- and N-terminus labels (and 5’ and 3’ labels) as superscripts in order to minimize confusion.

Reviewer Comment: In Figure 2, it would be helpful to include the PNA concentration in the caption.

Response: The caption now reads that the PNA and DNA are at a 1:1 ratio for all samples, with the x-axis of the graph showing the tested concentrations.

Reviewer Comment: Why use a 10 nucleotide overhang? My understanding of the literature is that the displacement rate maximizes at toehold length of around 5 nucleotides.

Response: Toehold systems rely on the masking sequence having higher affinity for the releasing sequence than the target sequence. Since the target is PNA and this forms duplexes with higher stability than DNA:DNA or DNA:RNA, we require a longer toehold in order to ensure that the MS:RS duplex represents a thermodynamically favorable state.

Reviewer Comment: Why place the mismatch at the 3’-end of the masking sequence when an internal mismatch is more likely to cause destabilization?

Response: We agree with the reviewer that introducing a mismatch in the middle of the sequence could also be interesting, but we were concerned that the impact on the hybridization stability and assembly properties would be too dramatic. We have added a brief rationale to the manuscript for our choice of mismatch location:

We chose to locate the mismatch at a terminal site rather than an internal site for two reasons. First, internal mismatches in PNA can have a significant impact on duplex stability and we recognized that this would likely prevent efficient hybridization at room temperature. Second, the primary role of the masking sequence is to shield the hydrophobic block of the PNA sequence, and thus we sought to locate the mismatch as far away as possible from this region of the duplex.

Reviewer Comment: Bizarre result #1: the authors find that the mismatch leads to higher PNA-DNA melting temperature. Good for the authors to include this and point it out to the readers, but it does leave one wondering why the Tm increased for a T-T mismatch.

Response: While this result was unexpected, the melting temperatures of the two systems were not significantly different from each other at 59 °C and 61 °C, respectively, for the matched and mismatched sequences. This is a minor difference and could be attributable to competition between hybridization and folding of the individual sequences. We chose to include these data out of transparency, and found that further optimization was not needed, as our matched system provided sufficient performance for disassembly and reassembly.

Reviewer Comment: I realize that RNA-DNA typically has lower Tm than DNA-DNA but the large difference in % Displacement for RNA vs DNA (Figure 3B) is surprising to me. Is this bad intuition on my part or is there some other explanation, such as the experiment being done close to the Tm for RNA-DNA, magnifying the affinity difference to a much larger degree than if the experiment were done well below both Tms?

Response: This is indeed the case, as the Tm of the PNA:MS and MS:RS duplexes are close to one another, and thus small changes in relative duplex stability lead to significant changes in the location of equilibrium.

Reviewer Comment: At the top of page 9, the authors state that differences in concentration, stoichiometry and buffer conditions could explain why they observe an opposite trend for RNA releasing sequences compared to predictive models. I can see how experimental differences might change the magnitude of an effect, but reversing it is harder to understand.

Response: While the effect of conditions such as ionic strength on hybridization for native nucleic acids is well understood, the effect on PNA duplexes is significantly more complex. Thus, while the reversal was unexpected, it is not entirely surprising.

Reviewer Comment: Bizarre result #2: In Figure 4C, why is there no effect on % Displacement of going from 1.0 to 1.5 equivalents of RS-R but then the displacement increased from 1.5-2.0 equivalents?

Response: This result is likely due to the larger error bar for the 1.5 eq samples. Taking into consideration the error, the displacement still follows an approximately linear trend.

Reviewer Comment: In Figure 6, PNA-A1-FAM is shown at 100 uM whereas the other samples are shown at 10 uM. It seems that PNA-A1-FAM alone should also be shown at 10 uM.

Response: We appreciate this feedback and have performed this experiment and updated Figure 7 accordingly.

Reviewer Comment: I don’t know what to make of the DLS experiments, which seem to contradict the central hypothesis of the work, namely that adding a masking strand would prevent aggregation when what is observed is a 10-fold increase in particle size. Moreover, addition of the displacing strand has no effect on the size. Rather than conclude that their approach doesn’t work, the authors instead decide that DLS isn’t appropriate for measuring the effects and resort to TEM. However, TEM only reflects what was deposited on the grid and in fact the grid and uranyl acetate stain could be influencing the equilibria. Also, the concentrations of the DLS and TEM experiments were quite different. I guess I’m left with doubts about whether this elegant design actually works.

Response: We contend that DLS did not provide conclusive results for the disassembled and reassembled samples. This was not entirely unexpected, given our previous observation of the formation of large non-specific aggregates upon addition of DNA or RNA to PNA samples, coupled with the fact that large assemblies can dominate the DLS signal, prohibiting detection of smaller assemblies. To provide greater clarity, we have moved these DLS results to the SI and revised the manuscript to better explain that the TEM results are what provide evidence for disassembly and reassembly. We highlight that this is not an attempt to dismiss the DLS data, but rather reflects a common challenge in DLS.

We do feel that the TEM data do demonstrate disassembly and stimuli-responsive reassembly. Specifically, while we observe large aggregates upon addition of DNA and RNA (similar to in our previous work), we also see the disappearance of the ordered PNA assemblies upon addition of masking strand, followed by reappearance of these assemblies upon addition of releasing strand. We have elaborated upon our discussion in the manuscript to highlight this point:

While we do still observe some non-specific aggregates, we were excited to also observe a large number of ordered assemblies having an average size of 16.8 ± 5.7 nm (Fig. 7C). We do note that the structures formed upon reassembly are slightly smaller than those formed in the initial PNA assembly. This may be due to the presence of DNA in the solutions or as a result of a different assembly mechanism when the PNA is being released from a DNA complement over time. However, we were excited to observe that our bilingual PNA is able to undergo stimuli-responsive assembly as anticipated.

19-May-2022

Dear Mr Argueta-Gonzalez:

Manuscript ID: CB-ART-01-2022-000020.R1
TITLE: Stimuli-Responsive Assembly of Bilingual Peptide Nucleic Acids

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 your manuscript and the reviewers’ reports, I will be pleased to accept 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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Institute of Organic Chemistry, University of Würzburg

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Reviewer 2

The authors have satisfactorily addressed my concerns from the original submission. Although the interpretation of the disassembling-reassembling may still be debatable, I think it can be published and let the readers decide.
However, a few minor corrections should still be made in the revision including:
- The description of Fig 5 is not clear. Three different sizes were mentioned (1.5, 80 and 119 nm), but only two peaks are shown.
- The stereochemistry of the gammaPNA in Fig. S7 may be incorrect. Should they be in the L-form (ie, with the substituents pointed down if drawn that way) rather than D-?

Reviewer 1

The revised manuscript is greatly improved.
I'm not convinced that the introduction of a mismatch more in the middle of the sequence will totally disturb the formation of the duplex at room temperature since the Tm of the fully matched duplex is around 60 °C or will notably change the hydrophobicity of the duplex.

Here are some suggestions to improve the manuscript.

Caption of figure 1; It will be interesting to define the nature of R1 and R2 to better understand why the resulting PNA form micelles.

For Fig 3C it is difficult to see that RS-R 1hr is similar to RS-D 1hr. It will be better to put the red circle on the black square, or induce a small x-offset to visualize the circle and the square.

The CD study is clear. However, as suggested at the end of page 18, (Fig 6) it will be interesting to perform the CD curves subtraction [PNA-A1-FAM:MS-D+RS-D] - [MS-D:RS-D] to see if it is close to the CD curve of PNA-A1-FAM alone, and perform the addition of [MS-D:RS-D] + [PNA-A1-FAM] and compare to the CD curve [PNA-A1-FAM:MS-D+RS-D]. This could give an evidence that the PNA-A1-FAM does not interact with the MS-D:RS-D duplex. Please comment the results.

After these minor modifications, the manuscript could be accepted for publication in RSC Chem Biol.

June 2, 2022



Prof. Dr. Claudia Höbartner
Associate Editor
RSC Chemical Biology

Dear Prof. Dr. Höbartner,

We would like to submit a revised version of the manuscript CB-ART-01-2022-000020 entitled “Stimuli-Responsive Assembly of Bilingual Peptide Nucleic Acids” for publication in RSC Chemical Biology. We appreciate the feedback from the reviewers and agree that clarification was needed. We have addressed these minor errors and editorial issues as requested.

This cover letter includes a detailed list of our responses and changes to the manuscript. We thank the reviewers for their feedback.

We appreciate the reviewers’ comments and your time in considering this modified version of the manuscript.

Sincerely,

Jennifer M. Heemstra
Professor
Emory University
1515 Dickey Drive
Atlanta, GA 30322
404-727-7766
jen.heemstra@emory.edu














RESPONSES TO REVIEWERS’ COMMENTS

REVIEWER 1
Reviewer Comment: The description of Fig 5 is not clear. Three different sizes were mentioned (1.5, 80 and 119 nm), but only two peaks are shown.

Response: We do observe a peak at 80 nm but this peak accounts for less than 1% of the distribution of objects and thus it is not visible in the graph in the manuscript. To avoid confusion, we removed mention of the 80 nm peak and instead indicated that only peaks representing >1% of the population are listed. We do still list this peak in the DLS graph in the Supporting Information.

Fig. 5 Normalized size distribution of PNA assemblies. Samples tested at 200 µM in 1x PBS. Average diameter of particles of PNA-C1-FAM = 1.5 ± 0.3 nm; PNA-A1-FAM = 119.1 ± 34.0 nm. Peaks having greater than 1% are reported.

Reviewer Comment: The stereochemistry of the gammaPNA in Fig. S7 may be incorrect. Should they be in the L-form (ie, with the substituents pointed down if drawn that way) rather than D-?

Response: We thank the reviewer for catching this error and we have updated Figure S7 with the correct stereochemistry.


REVIEWER 2

Reviewer Comment: Caption of figure 1; It will be interesting to define the nature of R1 and R2 to better understand why the resulting PNA form micelles.

Response: The figure caption has been updated to further elaborate on the design principle for the amphiphilic PNA sequences.

Fig. 1 (A) Bilingual PNAs having amphiphilic side chains at the γ-position to direct self-assembly and a nucleotide sequence to direct disassembly through recognition of a complementary masking sequence. The hydrophobic group (R1) drives self-assembly by inducing aggregation, while the hydrophilic group (R2) increases solubility. (B) Stimuli-responsive assembly through toehold-mediated displacement of the masking strand.

Reviewer Comment: For Fig 3C it is difficult to see that RS-R 1hr is similar to RS-D 1hr. It will be better to put the red circle on the black square, or induce a small x-offset to visualize the circle and the square.

Response: We thank the reviewer for this suggestion. The figure was modified to show the RS-R (red data points) over the RS-D (black data points).

Reviewer Comment: The CD study is clear. However, as suggested at the end of page 18, (Fig 6) it will be interesting to perform the CD curves subtraction [PNA-A1-FAM:MS-D+RS-D] - [MS-D:RS-D] to see if it is close to the CD curve of PNA-A1-FAM alone, and perform the addition of [MS-D:RS-D] + [PNA-A1-FAM] and compare to the CD curve [PNA-A1-FAM:MS-D+RS-D]. This could give an evidence that the PNA-A1-FAM does not interact with the MS-D:RS-D duplex. Please comment the results.

Response: We have performed the subtraction analysis as suggested by the reviewer and this has been added to the SI as Fig S14. The additional analysis further confirms that the PNA-A1-FAM is released from the PNA:DNA duplex, resulting in formation of a DNA:DNA duplex, as subtraction of the PNA-A1-FAM:MS-D+RS-D signal from the MS-D:RS-D signal matches the PNA-A1-FAM signal. We believe this additional analysis sufficiently confirms the release of the PNA-A1-FAM.

16-Jun-2022

Dear Mr Argueta-Gonzalez:

Manuscript ID: CB-ART-01-2022-000020.R2
TITLE: Stimuli-Responsive Assembly of Bilingual Peptide Nucleic Acids

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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Claudia Höbartner
Associate Editor, RSC Chemical Biology
Institute of Organic Chemistry, University of Würzburg


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