Active generation of nanoholes in DNA origami scaffolds for programmed catalysis in nanocavities

DNA origami tiles provide nanostructures for the spatial and temporal control of functional loads on the scaffolds. Here we introduce the active generation of nanoholes in the origami scaffolds using DNAzymes or light as triggers and present the programmed and switchable catalysis in the resulting nanocavities. We engineer “window” domains locked into the origami scaffolds by substrates of the Zn2+-ion- or Pb2+-ion-dependent DNAzymes. Using Zn2+ ions and/or Pb2+ ions, the programmed unlocking of the “window” domains is demonstrated. The tailored functionalization of the origami scaffolds allows the programmed operation of catalytic processes in the confined nanocavities. Also, the “window” domain is integrated into the origami scaffold using photoisomerizable azobenzene-modified locks. The cyclic photoisomerization of the locks between the cis and trans states leads to a reversible opening and closure of the nanoholes and to the cyclic light-induced switching of catalytic processes in the nanocavities.

1. While the AFM data and the corresponding statistics for the respective experiments were shown as a 4 panel figure, each panel being an image from a different scanned area, it would be interesting to know exactly which parameters/conditions these images differed in, i.e. for example are these 4 different scanned areas on the same mica surface i.e. the same replicate? Or are these from different replicates of the same experiment?
2. There is essentially no doubt regarding the results of all the experiments, in that there seem to be very clear differences between all the experiments and their corresponding control treatments. However it is just good scientific form to also show some preliminary statistic tests to accompany the claims.
Grammar and typo 1. L57 -"...chiroptical functions were demonstrated..." should be "chiroptical functions have been demonstrated" 2. L60 -"...functional origami structures included, however, the bottom-up..." should be "functional origami structures involved, however, the bottom-up" 3. L64 -"To date, such cavities were fabricated..." should be " To date, such cavities have been fabricated" 4. L65 -"...and the cavities were used for site-specific docking of antibodies, the reconstitution of membrane proteins..." should be "and these cavities were used for the site-specific docking of antibodies, reconstitution of membrane proteins..." 5. L67 -"In contrast to this previous art, the present study introduces a concept for the "active" fabrication of nanoholes in origami tiles." should be "In contrast, the present study introduces the concept of "active" fabrication of nanoholes in origami tiles." 6. L73 -"...we highlight the design of a reversible..." should be "we highlight a design for the reversible" 7. L294 -Typo -"nanohole-patters" should be "nanohole-patterns" 8. L 480 -Typo -"spectru" should be "spectrum" Reviewer #2: Remarks to the Author: The manuscript by Wang et al. presents an exquisite DNA origami tile featuring a switchable catalytically active cavity.
Letters, 2018, https://doi.org/10.1021/acs.nanolett.8b00793). In this earlier publication, the active state of the DNAzyme (catalyzing a similar reaction) was triggered by the dimerization of two DNA origami subunits instead to the opening of a cavity within the origami.
The authors have done a considerable amount of work to demonstrate the opening/closing of the cavity upon application of different stimuli, but several points should be addressed before publication in Nature Communications or elsewhere: Comments: 1) In the introduction, the authors state that their study represents the first example of an active generation of nanocavities. This is not true. 2) For a non-specialist reader, it would be beneficial if the authors could provide literature references when they first mention the used DNAzymes. More descriptive details on the catalytic reaction and the functioning of the DNAzyme could be helpful.
3 6) Given that AFM imaging is used as the key method throughout the manuscript, the authors provide very limited details about the imaging parameters in the method section. E.g. it remains unclear which cantilevers (spring constant?) were used. This section has to be expanded.
7) The authors compare the activity of the DNAzyme in closed and open DNA origami cavities. How do the values compare to the activity of the bare enzyme? Does cavity opening fully restore its activity?
8) The authors demonstrate an "on-off-on-off" cycle to showcase the reversibility of their system (Fig. 4). Have they tested or can they estimate how many repeats are possible? This should be discussed.

Response to referees
The following changes/explanation were introduced into the paper.
Reviewer #1: I appreciate very much the very positive evaluation of the reviewer and his/her comment that "… … the experiment simple and elegant in design, and the corresponding interpretations relatively concise, straight forwards and easy to understand. I honestly found it somewhat difficult to come up with any criticism".
Answers to the specific comments: 1. "While the AFM data and the corresponding statistics for the respective experiments were shown as a 4 panel figure, each panel being an image from a different scanned area, it would be interesting to know exactly which parameters/conditions these images differed in…" Reply: The issue of statistical analysis of the images was addressed in detail in the experimental section, heading AFM imaging, in the revised manuscript, page 20.

"… it is just good scientific form to also show some preliminary statistic tests to accompany the claims."
Reply: We added the statistical analysis of the control systems and these are outlined in new tables: Supplementary Table 2, Supplementary Table 3, Supplementary Table 5,  Supplementary Table 6, and Supplementary Table 15 and Table 16. These statistical analyses were mentioned in the revised manuscript, pages 6, 7 and 12, respectively.

"Grammar and typo"
Reply: All errors were corrected in the revised manuscript, pages 3, 4, 14 and 17, respectively. Reviewer #2: "A similar concept was presented by the authors in an earlier publication (Wang et al., Nano Letters, 2018, https://doi.org/10.1021. …" Unfortunately, we disagree with this statement of the reviewer. The quoted reference significantly differs from the present work. In the quoted reference, we described the interconversion between two origami tiles using the strand displacement process. The present study introduces the DNAzymes and light as unlocking mechanisms of a domain on the origami tile and the development of an active "window-opening" mechanism using "handles", "helper units" and anchoring footholds.
Answers to the specific comments: 1. "In the introduction, the authors state that their study represents the first example of an active generation of nanocavities. This is not true. …" Reply: The statement, although being correct, was not introduced into our original version of the paper. The references mentioned by the reviewer have little relevance to the present paper. They describe the incorporation of nucleic acids into membranes and the generation of nanochannels for transport. These references do not deal with DNA origami structures. To follow the reviewer comment, we introduced the two references to the introduction and explained the differences between our work and the stated references in the revised manuscript, page 3.