Review Reports - Recognition Mechanism of Complementary Nucleobases and Sequences in DNA and RNA: Interplay of Watson–Crick Hydrogen Bond Formation and Base Stacking Interactions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review paper by Takahashi and Norden entitled “Recognition of complementary base sequences” provides an overview of biological processes and applications involving base-pair formation, and summarizes the molecular mechanisms underlying complementary base recognition.

This topic is important, and the manuscript is generally well written. However, I would like to request that the authors respond to the following comments. Substantial revision is required for this manuscript. In order for the manuscript to be accepted, the authors must address all of the comments below and improve the manuscript accordingly.

(1) Please correct line 5 as follows:

Takahashi1^{1, 2, *}and → Takahashi^{1, 2, *}and

(2) Please correct “Hirata” to “Hirao” in lines 310 and 319.

(3) In lines 321 and 322, the phrase “that were correctly replicated in vitro and in vivo.” is used. However, there are no successful examples in vivo. Please revise this statement accordingly.

(4) Please correct the error in the chemical structure of the nucleobase dF shown in Figure 3.

(5) In line 255, the manuscript states “despite the absence of hydrogen bonding.” However, it is already well known that this effect arises from shape complementarity (shape fitting). Please add an appropriate comment to clarify this point.

In addition, to help readers easily understand this concept from the figure, CPK models should be used in Figure 3a, as shown in Figures 4a and 4b of the following paper:

Proc Jpn Acad Ser B Phys Biol Sci. 2012;88(7):345 (DOI: 10.2183/pjab.88.345)

(6) The authors state that “This review provides an overview of biological processes and applications involving base pairing and discusses the molecular mechanism underlying complementary base recognition.”

If this is the case, structure-based studies, such as those using X-ray crystallography or NMR spectroscopy, should be cited and discussed in more depth.

For example, in the context of Hirao’s work, papers such as DOI: 10.1002/anie.201704190 should be cited and discussed.

(7) In Section 4.4, the authors describe Hirao’s work; however, the final sentence cites Ref. 62, which corresponds to a study by Kool et al. This is confusing. Please correct the citation to clearly distinguish between these studies.

In addition, Hirao and coworkers have successfully obtained aptamers using their artificial base pairs. Shouldn’t these studies also be cited (e.g., DOI: 10.1038/nbt.2556 and DOI: 10.1021/jacs.6b10767)?

(8) Although the manuscript mentions that Hirao and coworkers obtained aptamers using artificial bases without hydrogen bonding, it does not appear to discuss the work of Steven A. Benner and coworkers, who have obtained aptamers using artificial bases that do form hydrogen bonds.

Isn’t this an essential point when discussing the molecular mechanisms of complementary base recognition?

(9) In Section 5.1, the authors discuss kinetic analyses, but should thermodynamic analyses not also be discussed? Thermodynamic considerations are essential when discussing the molecular mechanisms underlying complementary base recognition.

Author Response

Combine figures into a single large figures for better readability, especially since individual figures are very small by themselves.

Our response: We agree with the Reviewer that the figure material should be displayed at a magnification that makes them individually easy to clearly read. We hope the copy-editor could help us display the figures in a proper way – for example Figure 1 and 2 printed side-by-side if page size allows that. At the same time, just as the Reviewer says, the figures must be clearly readable and, therefore, cannot be decreased significantly in size. We leave the figures as they are in the Word document and await the copy-editor’s suggestions.

While paper highlights basepair mechanisms in CRISPR, RNA silencing, etc, no specifics are provided into techniques that are commonly used to study such mechanisms (e.g FRET, stopflow, cryoEM, x-ray, MD simulations, etc). The authurs could put some more emphasis on highlighting techniques that enable understanding such mechanisms.

Our response: We thank the Reviewer for this valuable comment which made us check, and when needed complement, references so that techniques are appropriately referred to when studying base-pairing mechanisms. For example, FRET is abundantly used to follow DNA exchange dynamics. To follow the Reviewer’s suggestion a whole section referring to the adequate structural and kinetic techniques as applied in recent investigations of base-pairing has been introduced (line 445-471) together with appropriate literature references (Refs 68-74).

Page 1, line 36 - The line should read DNA "comprises" two strands.. Our response: Typo has been amended.
Page 4, line 129 - "DNA polymerase makes only one mistake over 107 events." This statement seems incorrect or likely typo. Can the author provide some ref, if correct? Our response: This is a typo (10⁷) but the fact that it varies (as can be found in any textbook) has made us insert ‘typically’.

5.page 12, line 444 - "Understanding the mechanism is required for efficient applications." This statement feel out of place. Can the authors clarify what do they mean by reference to "efficient applications"? Our response: We are grateful to the Reviewer for discovering this inconsistency. The whole paragraph has been rewritten to clarify the point of Conclusions.

Comments on the Quality of English Language

Some lines are very long and hard to read. The authors could break down some of the lines to split into 2 sentences for better readability. Overall, quality of english is good. Our response: The whole paper has been given a careful linguistic overhaul.

Reviewer 2 Report

Comments and Suggestions for Authors

The review article from Takahashi and Norden presents a unique insight into basepair interaction and complimentarity, driven by WC hydrogen bonding and base stacking interaction. The content of this review is comprehensive to understaning of these molecular mechanisms, describing various biological processes in living organisms. Ovell the article is well written and I have no major critics. I have a few suggestions to make this more helpful for broader audience.

Combine figures into a single large figures for better readability, especially since individual figures are very small by themselves.

2. While paper highlights basepair mechanisms in CRISPR, RNA silencing, etc, no specifics are provided into techniques that are commonly used to study such mechanisms (e.g FRET, stopflow, cryoEM, x-ray, MD simulations, etc). The authurs could put some more emphasis on highlighting techniques that enable understanding such mechanisms.

3. Page 1, line 36 - The line should read DNA "comprises" two strands...

4. Page 4, line 129 - "DNA polymerase makes only one mistake over 107 events." This statement seems incorrect or likely typo. Can the author provide some ref, if correct?

Comments on the Quality of English Language

Some lines are very long and hard to read. The authors could break down some of the lines to split into 2 sentences for better readability. Overall, quality of english is good.