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Peer-Review Record

Synthesis of Sensitive Oligodeoxynucleotides Containing Acylated Cytosine, Adenine, and Guanine Nucleobases

by Komal Chillar, Rohith Awasthy, Marina Tanasova and Shiyue Fang *
Reviewer 2: Anonymous
Submission received: 24 February 2025 / Revised: 9 March 2025 / Accepted: 16 March 2025 / Published: 9 May 2025

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I have reviewed the manuscript titled "Synthesis of sensitive oligodeoxyribonucleotides containing acylated cytosine, adenine, and guanine nucleobases." You have successfully applied your previously reported protocol to synthesize sensitive ODNs incorporating 6acA, 2acG, 4mcC, and multiple 4acC modified nucleotides using Dmoc and meDmoc groups.

The manuscript is well-written and concise, effectively highlighting the work, particularly the incorporation of four 4acC modifications in their ODN. I appreciate that you also acknowledge limitations, such as the appearance of a shoulder in CE data and the absence of such modifications in nature. I recommend the publication of this manuscript in "DNA" if you can address the following queries:

1. Given that these modified nucleotides are not reported in nature, please provide a rationale for their synthesis. I suggest conducting basic characterization studies, such as UV absorption spectroscopy of both single-stranded and base-paired double-stranded ODNs.
    
2. While Fig. S1 shows a denaturing PAGE gel of these ODNs, please include a native PAGE gel of base-paired dsODNs to assess the hybridization ability of modified nucleotide-containing ODNs compared to their canonical counterparts.
    
3. Clarify whether four 4acC modifications represent the maximum attempted. If so, explain the reasoning behind this limit.
    
4. Provide the rationale for choosing an ODN length of 19 nucleotides.
    
5. Explore and discuss the limitations of the methodology in terms of:  
    a. The maximum number of modifications that can be stably incorporated into a single ODN.  
    b. The feasibility of incorporating all the four different modifications demonstrated in this work within a single ODN.
    
6. Present data on the coupling efficiency for each modification compared to unmodified base coupling.
    
7. Comment on the shelf-life of these modified ODNs relative to their unmodified counterparts.
    
8. Explain the choice of the Dmoc and meDmoc method. If other synthesis methods were attempted, please include a brief discussion of unsuccessful attempts to benefit early-career scientists.
    

Addressing these points will strengthen the manuscript and provide valuable insights for readers. I look forward to reviewing your revised version.

Sincerely,
Vinod

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper by Chillar et al. reports ultra-mild DNA syntheses for modified oligonucleotides (ONs) with acylated cytosine, adenine, and guanine nucleobases. The authors prepared phosphoramidite monomers bearing N4-methoxycarboyl-dC, N6-acetyl-dA, N2-acetyl-dG, together with commercially available N4-acetyl-dC to synthesize ONs with acetyl groups. Since these groups are all base-labile, they adopted methyl 1,3-dithian-2-yl-methoxycarbonyl (meDmoc) linker to tether first nucleoside unit to the CPG support, replacing dimethoxytrityl (DMT) group with trityl (Tyr) group for the protection of 5'-OH, which enables two-step cleavage from the support and ultramild deprotection of resulting ONs with non-nucleophilic K2CO3. The authors successfully confirmed that all of the three base-labile acyl-groups on nucleobases are intact in the cleavage and deprotection process, giving slight shouldered HPLC peak but sharp single peaks in capillary electrophoresis and MALDI-TOF/MS.

  Experimental setup and results look convincing, and the logical flow of the manuscript is reasonable. I recommend acceptation for publication after the following minor points are addressed:

  1. Term "sensitive": I think "base-labile" is the most standard term to represent the "sensitivity" of protective groups in organic chemistry as well as in nucleic acid chemistry. I recommend replacing at least the one found in the Abstract (line 8) for easier understanding of the concept by readers in the field.
  2. Full spelling of "meDmoc" and "Dmoc" should be prsented in the Abstract (lines 11 and 12).
  3. Figure 2: the lower part of the "Figure" showing the pathway from compound 3 to 6 is a reaction "Scheme" in fact. I recommend to add "Scheme 1" for the part.

Author Response

Please see attachment

Author Response File: Author Response.pdf

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