High Bendability of Short RNA-DNA Hybrid Duplex Revealed by Single-Molecule Cyclization and Molecular Dynamics Simulations
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript will be acceptable after minor revision.
1, The authors described that the RDH duplexes are anchored to the polymer-coated glass surface to avoid dimer formation. Despite being immobilization, dimer would be formed when the RDH duplexes are anchored in the high density. How much of the density do the authors estimate?
2, I would like the authors to explain the reasons why high concentrations of Na+ can stabilize the looped state.
3, I I would like the authors to discuss the reasons why C-rich DNA and G-rich RNA shows higher flexibility, based on the chemical structures of bases.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript “Extreme Bendability of Short RNA-DNA Hybrid Duplex Revealed by Single-Molecule Cyclization and Molecular Dynamics Simulations” by Xinghua Zhang et al. combines single-molecule cyclization experiments and molecular dynamics (MD) simulations to demonstrate that short RNA-DNA hybrid (RDH) duplexes exhibit significantly greater bendability than DNA duplexes of the same sequence. The authors further show that RDH duplexes composed of C- or G-rich NA are particularly flexible, suggesting a potential biophysical basis for R-loop formation in GC-skewed regions. The manuscript is logically structured and well-organized, which aids reader comprehension. Methodologically, the work is sound and provides valuable insights into RDH mechanics. The sequence dependence of RDH flexibility, especially within the context of GC skew of APOE gene promoter is a novel and relevant finding.
However, the conclusions are currently based on a limited number of sequences from the APOE. Additional data would be needed to validate the generality of these findings.
The use of smFRET-based cyclization assays is appropriate and effective for probing short-range nucleic acid flexibility.
However, the application of MD simulations in this DNA/RNA context raises some concerns. The OL21 force field is optimized for DNA, not RNA, and may not accurately model hybrid structures. A combination of force fields (e.g., OL21/OL24 for DNA and OL3 for RNA) might be more appropriate. It is also unclear why pre-formed RDH constructs were used instead of simulating hybridization explicitly.
The MD results should be further validated using positive and negative controls and by intentionally altering force field parameters to assess the robustness of the findings.
The MD setup/FF does not suggest any opening of the dsDNA, or at the very least, such an event appears energetically disfavored.
Despite these issues, the experimental analysis is strong. Looping kinetics are clearly analyzed, and the correlation between experimental looping times and MD-derived structural parameters is convincing.
The manuscript merits minor revisions, although additional analyses—particularly of MD robustness and sequence diversity—are strongly recommended. Even in its current form, the study offers new and interesting insights, is clearly written, and was, in all honesty, a pleasure to read. Please Adjust the conclusions. And check the title “extreme bendability” may suggest supercoiling?
Major Issues
Only a few sequences from the APOE promoter were analyzed. Testing additional regions with varying GC skew would strengthen the claims of sequence-dependent flexibility.
While R-loop processing proteins (RNase H) are discussed, they are not experimentally investigated. Including either experimental or coarse-grained simulation data on protein interactions would add significant value.
The assertion that RDH bendability drives R-loop formation should be made more cautiously. Other contributing factors - such as G4 formation, DNA supercoiling (positive or/and negative), and protein-DNA interactions - are not accounted for and could play significant roles.
minor suggestions
abstract> ... and is more easily to form kinks than DNA >> and more easily forms kinks than DNA ... ?
L410 could contributed >> could contribute
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
