I am thankful to Prof. Rebecca Schulman and Dr. Yi Li for in-depth review of this manuscript and sincerely appreciate their insightful comments. I have revised the manuscript as per these comments. I sincerely believe that the reviewers will find the revised manuscript suitable for their approval. The point-by-point response to the reviewer’s comments is given below.

Comment: Plasmid DNA molecules have been determined by researchers to be circular, double stranded helical structures. In this work, however, the author claims plasmid DNA molecules follow a non-helical model, supported by experimental results that showed alkali-denatured plasmid DNA is not intertwined but separated. The author further showed separated single stranded plasmid DNA renatured to form native plasmid DNA at neutral pH that could transform E. coli bacteria.

This article challenges the helical DNA model of plasmid DNA accepted by the mainstream scientific community. However, the claim that plasmid DNA is not intertwined cannot be supported by the experimental results presented and the logic in the article is flawed.

The agarose gel electrophoresis was the only method used to show separation of plasmid DNA strands. Figures in the article showed that alkali-denature plasmid DNA resulted in a band of higher mobility, which could only be digested by S1 nuclease that degraded single stranded DNA. However, the appearance of a higher-mobility band could not prove that the two single strands of were separated and not intertwined. It was totally possible that the denatured DNA strands did not form helices but still had a part intertwined to each other to keep them from separating. Such intertwined DNA could also be digested by S1 nuclease but not Hind III or exonuclease. Furthermore, since the two strands might not be separated, they could easily re-hybridize to form native plasmid DNA in the neutralized pH. Thus, the author’s claim that the two strands of the plasmid were separated was untenable.

The helical structure of plasmid DNA has been supported by many studies, some of which also used denatured plasmid DNA and presented results directed contradicted to the claims of this article. JiaYu et al. showed alkali-denatured plasmid could either remain supercoiled double-stranded or became single-stranded but not separated with atomic force microscopy, both of which showed increased mobility in agarose gel. Lifeng Yan and Hiroshi Iwasaki showed similar structures with AFM after thermal denaturation. The phenomenon that the two strands of plasmid DNA could not be separated under denaturing conditions has been demonstrated with electron microscopy and other techniques decades ago. The author should comment on those papers in this article when proposing a contradicting model.

Response: I appreciate the reviewer’s point regarding the possibility that the denatured DNA strands had a part intertwined with each other to keep them from separating. To rule out this, higher electrophoretic mobility DNA (HmDNA) in NaOH-denatured plasmid was compared with the individual strands of plasmid DNA for its electrophoretic migration in the gel. Interestingly, HmDNA and the individual (top and bottom) strands of pCMV6-AN-HA exhibited comparable electrophoretic mobility. These results corroborated the single-stranded and circular nature of HmDNA and have been incorporated in the revised manuscript (figure 1f).

Further, the reviewers have cited some studies to counter the results presented in this manuscript. However, I wish to highlight that in Jia Yu and colleague’s study, the authors had extracted alkali-denatured plasmid from gel before examining it by atomic force microscopy [1]. It is noteworthy that during gel-extraction procedure, alkali-denatured DNA reanneals into double-stranded DNA molecule (please see figure 2b in our manuscript). In keeping with this, no significant difference is seen between ‘form I’ and ‘form IV’ DNA in Jia Yu’s study except that the ‘form IV’ DNA image is slightly blurred (please see fig. 2).

In Lifeng Yan’s study, the authors denatured plasmid DNA by heating its solution [2]. However, it should be noted that heating is not a very efficient method to denature plasmid DNA. Xiaofang Wang et al. have shown that heating DNA fragments at 95°C for up to 30 min did not denature the DNA fragment [3]. Similarly, Stroop et al. demonstrated that heating pBR322 in boiling water for up to 300 seconds led to degradation, but not the denaturation of plasmid DNA [4]. It is also noteworthy that Yan et al. did not confirm the denaturation of plasmid DNA before observing it by Atomic force microscopy.

In other studies too, the authors had denatured plasmid DNA by heating DNA solution. However, the word limit of the present article prohibits discussion of these studies. Nevertheless, the new data in the revised manuscript confirm the argument that two strands of plasmid DNA are not intertwined with each other and therefore can be reversibly separated into single-stranded circular DNA molecules.

References:

Yu, J., et al., Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy. Biochem Biophys Res Commun, 2008. 374(3): p. 415-8.

I am thankful to Dr. Bharathwaj Sathyamoorthy and Dr. Abhishek Cukkemane for reviewing this manuscript and appreciate their insightful comments. I have revised the manuscript as per these comments. I sincerely believe that the reviewers will find the revised manuscript suitable for their approval. The point-by-point response to the reviewer’s comments is given below.

Comment: A common methodology that is employed during plasmid preparation is denaturation by NaOH followed by renaturation with acetic acid. The experiments performed in this article seem to be reproducing the same and is on expected lines. The reference of a basic textbook such as TA Brown's Gene Cloning should be provided.

Response: It is to be brought to the reviewer’s kind attention that a previous article, wherein plasmid DNA was isolated by the alkaline lysis method has been cited in the manuscript.

Comment: Enzymatic digestions performed in this study are done in the presence of NaOH in a number of cases, where the pH of the solution would be ~13 going by the concentrations given by the author. At this pH will the enzymes HindIII, S1 nuclease and Exo1+AP function at all? Since, the bands observed in these gels in the presence of NaOH is used as an observation to support the hypothesis of the study. This is a concern that must be addressed by the author.

Response: My apology for this confusion. Indeed, the enzymes used in this study would not work in the presence of 0.5 N NaOH (as also observed in our results, lane 5 in each of figure 1c-e). Therefore, for enzymatic digestion, NaOH was first neutralized using HCl (lane 3, pUC19+NaOH+HCl and lane 6, pUC19+NaOH+HCl followed by enzymatic digestion). The same is also stated in the methods section.

Comment: In Fig 1d, the author claims that S1 nuclease digests single stranded DNA in the right most lane (7th from left, 1st lane ladder) while double-stranded remains intact. Going by this argument, why hasn't the enzyme chewed up the NaOH denatured DNA (hmP19) band in the 6th lane?

Response: S1 nuclease did not digest single-stranded DNA in lane 6, figure 1d (lane 5 in the new version of article) because of the presence of NaOH. In lane 7 (lane 6 in the new version of article), NaOH was neutralized using HCl. Therefore, enzyme digested single-stranded DNA in this lane.

Comment: Additional point is that, the gel isn't capable of providing the information of what is the nature of the hmP19 band. It could very well be that the tertiary structure of the puC19 is altered in the presence of NaOH with Watson-Crick pairs remaining to a significant extent forming something like a super-supercoiled state of DNA. The assumption that hmP19 is a single stranded state seems a little far fetched with only gel based assays.

Response: It is indeed a very important point raised by the reviewers. To examine whether hmP19 DNA (HmDNA in the new version) is single-stranded circular DNA, its electrophoretic mobility was compared with that of purified strands of plasmid DNA. It was observed that HmDNA and individual (top and bottom) strands of pCMV6-AN-HA exhibited comparable mobility in agarose gel. These results corroborate the single-stranded and circular nature of HmDNA and have been incorporated in the revised manuscript (figure 1f).

Comment: One thing that I am unable to wrap around is that, if the two single strands are indeed not paired by Watson-Crick rules, then what is the author suggestion regarding the re-annealed DNA? How are they bound together? In similar line, it would help if the author gives some plausible models on what could hold the strands together that might pave way to more experiments.

Response: The main objective of the proposed study was to examine whether DNA strands are intertwined with each other as proposed by Watson and Crick. Interestingly, this study shows that two strands of a DNA molecule can be separated reversibly, contradicting the Watson and Crick model of DNA structure. Further, these results provide evidence for the side-by-side model of DNA structure. The same has been stated in the concluding paragraph of the manuscript.

I am thankful to Prof. Dmytro M Hovorun for timely reviewing my manuscript entitled “Non-intertwined strands of plasmid DNA contradicts the Watson and Crick model of DNA structure” and for his valuable comments.

I have carefully gone through Prof. Hovorun’s comments and found them both encouraging and insightful.  Although Prof. Hovorun appreciated the findings as quite interesting, one of his main concerns was regarding the usage of only a single technique (gel electrophoresis) in this study. Further, he suggested that additional arguments should have been provided using other techniques such as atomic force microscopy or vibrational spectroscopy.

I fully agree that the findings presented in this manuscript have been obtained using electrophoresis as the main readout technique. However, I would also like to draw Prof. Hovorun’s kind attention to the fact that owing to the above reason, this manuscript has been presented as a “Research Note”. According to the journal policy, Research Notes are small, often preliminary studies, descriptions of unexpected and perhaps unexplained observations or lab protocols that can be described in a short report with a few illustrations (figures/tables), or even a single figure.

The findings presented in this manuscript are in contradiction with the Watson and Crick model and are likely to have a huge impact on our understanding of DNA structure. I sincerely appreciate that these findings should be confirmed using other tools/techniques. I would also like to bring it to your notice that I am working in this direction and have obtained the encouraging results. I hope to publish the new findings very soon.