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  • Mathis Janßen1,*,
  • Anastasiia D. Murkina2 and
  • Julia Hann1
  • et al.

Reviewer 1: Xingyi Ma Reviewer 2: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript review

 

A Methodology for Validation of DNA origami – Quantum Dot Hybridization

 

Recommendation: Reconsider after revision and self-evaluation of the manuscript significance

 

This manuscript discusses a method for the confirmation of DNA origami-Quantum Dot hybridization. The authors argue that the proposed method based on FRET photoluminescence can be used for the confirmation of the annealing of DNA origami with other nanoobjects, such as nanoparticles and QDs. Smart methods for the characterization and validation of DNA nanomaterial functionalization are greatly significant, which makes this topic highly relevant and interesting to the scientific community. Additionally, the manuscript is well written with pertinent descriptions and analysis of the corresponding studies involved.

 

However, currently, there are sufficient methods to validate the functionalization of DNA nanomaterials with functional elements, such as QDs and nanoparticles. These characterization techniques include the as mentioned studies: gel electrophoresis and AFM as well as other methods such as DLS and Zeta potential analysis. Moreover, the proposed FRET-fluorophore-quencher validation method cannot be applied for the validation of several nanomaterials as FRET presents a distance-related limitation i.e., both the fluorophore and quencher must be placed in close proximity causing practical addressable issues. Therefore, the proposed method may not in practical terms solve the current bottlenecks especially considering that there are several more convenient methods for validating DNA origami functionalization.

 

Therefore, I would recommend it for publication after major revision of its content and perspective. Below, I have provided comments that the authors should take into consideration to improve this manuscript and strengthen their work significance, as many readers will rely on this work to improve their works.

 

  1. The authors proposed the use of FRET photoluminescence for the validation of DNA origami-QDs hybridization. However, currently validation techniques are satisfactory to the readers as well as top leading researchers in DNA nanotechnology field. Therefore, the authors are requested to:
  2. Find a stronger significance of their study as the current proposed method does not address a pressing problem in the field.
  3. Clearly specify in their manuscript whether the proposed FRET method can be applied to all studies of DNA origami functionalization or will the method inherit the limitation of FRET spectroscopic methods.

 

  1. In Section D. the researchers are recommended to promptly purify the QDs functionalized DNA origami and store the purified material at 4°C for further utilization.

 

  1. The authors are recommended (not mandatory) to add DLS data to strengthen the validation of the origami and origami-QD complex, if applicable.

 

  1. The authors are advised to use appropriate writing style throughout the manuscript and avoid terminologies and phrases such as the following:
  2. The bid advantage (abstract line 20)
  3. To get rid of (abstract line 23)

 

  1. Page 2 line 45 – 49. This section is highly redundant. The authors are advised to restructure the content concisely.

Author Response

Thank you very much for your thorough review. Please see the attachment as well as the revised manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

DNA origami (DO) technology has enabled precise nanoscale design through programmable self-assembly. Although the integration of functional entities such as nanoparticles, quantum dots, and biomolecules has greatly expanded the scope of DO-based systems, practical implementation remains limited by challenges in reproducible fabrication and purification. In particular, standard purification techniques like spin-column or membrane filtration often lead to aggregation and structural degradation of functionalized DO constructs, compromising yield and functionality. These limitations underscore the need for optimized, material-specific purification and validation strategies to ensure structural integrity and hybridization efficiency. Addressing this gap, the present work by Janßen et al proposes a refined workflow for producing and verifying quantum-dot–functionalized DO structures, employing a FRET-based fluorophore–quencher system and photoluminescence analysis as robust tools for assessing hybridization fidelity. Overall, the study and methodological designs convince to show potential strengths of the use of a FRET-based fluorophore–quencher system, combined with photoluminescence spectroscopy, enabling reliable confirmation of hybridization efficiency. However, there are certain sections which need to be strengthened for making the article articulate, before being accepted to the Applied Nano. Here are my comments on the article.

  1. The “Introduction” section is too short lacking several important background. Please explain the prevalent techniques used for DO characterizations (AFM, optical tweezers, magnetic tweezers, electron microscopies), and purifications (Amicon filtrations, PEG purification, etc.). Also, no background information on the properties of quantum dots is presented.
  2. Line 77: What kind of external forces were applied, please elaborate.
  3. How were the DNA sequences pair selected and finalized for Pair 1 and Pair 2. Was it based on their annealing temperature, free energy or anything else? Please explain in detail the rationale of selection.
  4. Line 120: Please mention the unit of averaging time. Was it 0.2 s, 0.2 ms or 0.2 µs?
  5. Line 139: I believe “incubation” would be a better word than “storage”. Also, it would be better to consistently use “1˚C/min” instead of “1K/min” for the rates.
  6. Lines 245-247: This needs to be written more scientifically mentioning the hybridized sequences have higher molecular weight compared to scaffold, which in turn slows the migration rate of conjugated samples.
  7. Since all the samples are at 10 nM concentrations, it is redundant to include 10 nM on the names in Table 1 and Figure 5.
  8. Conclusion section needs to be expanded, mentioning the limitations of proposed methods. How does the cross-talk between fluorophores affect results? What is the current resolution to observe the correctness/validation of origami structure? Does a single base pair irregularity/unhybridization affect the results for characterization of improperly synthesized origami? Also, please elaborate on the advantages and disadvantage compared to existing methods of DNA-PAINT and STORM.
  9. Please recheck the references. I am not sure why each articles have “und” before last authors and “Bd.” for edition.

Author Response

Thank you very much for your thorough review. Please see the attachment as well as the revised manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I am satisfied with the current version of the manuscript. The authors have revised it meticulously. Great job!