The Presence of Excitons in Short Single-Stranded DNA Revealed by Absorption and Circular Dichroism Spectroscopy
, Amy L. Stevens 2,4, Valentyn I. Prokhorenko 2 and R. J. Dwayne Miller 5,*
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsA brief Summary
This study utilized absorption and circular dichroism spectroscopy to investigate how the number and type of nucleobases in short single strands of DNA are related to excitonic coupling or dipole strength. It demonstrated that the degree, intensity, and presence of excitonic behavior can be identified, and it has the strength of experimentally confirming that these findings can be extended and applied to DNA and RNA of any length and sequence, provided that the molecules remain single-stranded and exhibit similar secondary structures.
1. The analysis of absorption and circular dichroism spectra to elucidate the extent of excitonic behavior and the relationship between dipole strength and the number of nucleobases in DNA single strands is indeed fascinating. However, there are some methodological concerns that warrant attention. Gaussian decomposition fitting was employed to analyze the absorption peaks, and the results were used to establish a relationship between dipole strength and nucleobase count. Since the experimental data itself is not explicitly presented, it raises questions about whether the Gaussian fitting truly represents the experimental spectrum. Furthermore, relying solely on these results to assert a linear relationship may be problematic. It could be more beneficial to explore alternative fitting methods, such as Lorentzian fitting or a combined approach using the Voigt function, to provide a more comprehensive analysis.
Additionally, a specific concern arises with the analysis of (dAT)n, where four Gaussians were utilized instead of three, as was the case for other sequences. While this choice may have been made to achieve a more accurate fitting outcome, comparing results obtained under different fitting conditions could potentially undermine the validity of the conclusions. Including supplementary materials with spectral plots or tables showing how the spectra vary with different numbers of Gaussians might help address these concerns.
In summary:
1) Have alternative spectral analysis methods, such as Lorentzian fitting or the Voigt function, been employed alongside Gaussian fitting?
2) Given the use of different numbers of Gaussians in the fittings, it would be helpful to provide spectral plots or tables showing how the spectra change with varying numbers of Gaussians to address any concerns about comparability.
2. The study appears to focus on characterizing the properties of short single-strand DNA. The experiments were conducted exclusively with AT base pairs, likely due to the practical ease of working with AT sequences and the availability of numerous references for comparison. However, one may wonder whether similar results would be observed with CG base pairs. If comparable findings can be demonstrated with CG sequences, it would strengthen the case for extending the applicability of this research to more general DNA structures, thereby enhancing the broader significance of the study. I don't necessarily think it's essential to conduct the experiment, but I would like to have some basis to predict whether the results would be similar to those of AT base pairs or not.
Minor Issues
3. In Figure 2 of the manuscript, the y-axis label for the CD spectrum differs, and the x-axis scale is inconsistent. Standardizing these elements is advisable.
4. Adding a legend to Figure 6 would improve clarity.
5. In Figure 7, it appears that changes in the CD spectrum are shown for two peaks across different families. Since the manuscript does not discuss the shorter wavelength peak, it might be more appropriate to retain only the longer wavelength peak.
6. The references to Burin's works (16 and 20) appear to be used inconsistently throughout the manuscript. For example, in Line 422, the experimental results for A-poly-T sequences correspond to Reference 16, yet Reference 20 is cited in Line 420 regarding the experimental results. Given the frequent citation of these two papers, it would be prudent to verify their use throughout the manuscript to avoid confusion.
Comments for author File: 
Comments.pdf
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThis article reported the absorption and circular dichroism (CD) spectroscopy study of short single stranded DNA (ssDNA) composed of 2 to 20 adenine (A), Thymine (T) or AT repeats. The goal of this study is to detect interactions between stacked nucleobases in ssDNA which predominately form H-aggregate and lack the contribution of the Watson-Crick hydrogen bond present in double-stranded DNA. The blue-shifted hypochromic absorption spectra and butterfly-shaped CD spectra suggest that the excitonic characters are present in the ssDNA samples. Data analysis showed that the extent of the excitonic coupling is limited to adjacent nucleobases for A whereas for T it spans multiple nucleobases and AT shows a mixed behavior.
I think that this study is an important step forward to understand the interactions between the DNA nucleobases. They did a great job explaining each and every feature of the all the plots with supporting of the existing literature.
In my opinion this paper would be suitable for Spectroscopy Journal after minor revision. The authors need to address few minor issues to make it more impactful.
1. Line 192-194: "The exciton delocalized between the bases along the strand, evidenced by the existence of a blue shift, was previously proposed by Hu (2);" - In the case of exciton delocalization one would expect to get a redshifted and two-peak-splitted absorption spectra. Why do we observe a blue-shift here?
My guess would be that there are two peaks present (CD spectra suggesting at ~280 nm and ~250 nm) and at room temp it is appearing as one broad peak.
2. Line 238 - "Due to the constancy of the shift, this may occur only at a few sites, but the broad nature of the absorption peak limits the available information." and Figure 3.
As concluded in the paper that the excitonic coupling spans multiple nucleobases, one would expect to see a further blue shift with increase in the number of nucleobases. Can you explain in detail why all the (dT)n ssDNA has the same absorption at 260 nm?
Maybe absorption spectra need to be collected at lower temp to see the difference in absorption spectra ~ 260 nm for (dT)n ssDNA.
3. Line 261 - "shown in Fig. Error! Reference source not found." - I believe it is a typo and it will be ... Fig.4.
4. Please add legend in Figure 6. The plot was explained in the figure caption, but it would be great if you can add the legend too. All the other figures have their legend.
5. In Figure 6, one may think that y-axis has value of First Gaussian m2 or Second Gaussian m2. But it is taking the value of (First Gaussian m2 + Second Gaussian m2). I could not find anything that mention that. Can you please update this in the caption?
Also, I was wondering why (dA)n has higher slope than that of the (dT)n plot.
6. Figure 14 is blocking some of the caption of Figure 13. Please place the Figure 14 accordingly.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsA brief Summary
As mentioned in the previous review, the analysis of absorption and circular dichroism spectra to examine how the number and type of nucleobases in short single-stranded DNA are related to exciton coupling or dipole strength is very intriguing. Additionally, the authors have provided sufficient explanations for the points that the reviewer questioned in the first review, making the paper easier to understand. Consequently, the findings of this study experimentally confirm that, as long as the molecule maintains a single-stranded structure and exhibits a similar secondary structure, these discoveries can be extended and applied to DNA and RNA of all lengths and sequences.
Comments and Suggestions for Authors
First of all, I would like to express my gratitude for kindly addressing the questions I had. I have thoroughly reviewed all the data in the main text, including the supporting information, and was able to confirm all the spectra without any confusion. You have also carefully verified the alternative fitting methods that were mentioned. By referring to the references you provided, I have confirmed the validity of the current analysis method. Additionally, I appreciate that you incorporated this information into the manuscript.
Comments for author File: Comments.pdf
