Integration of photocatalytic and dark-operating catalytic biomimetic transformations through DNA-based constitutional dynamic networks

Nucleic acid-based constitutional dynamic networks (CDNs) have recently emerged as versatile tools to control a variety of catalytic processes. A key challenge in the application of these systems is achieving intercommunication between different CDNs to mimic the complex interlinked networks found in cellular biology. In particular, the possibility to interface photochemical ‘energy-harvesting’ processes with dark-operating ‘metabolic’ processes, in a similar way to plants, represents an up to now unexplored yet enticing research direction. The present study introduces two CDNs that allow the intercommunication of photocatalytic and dark-operating catalytic functions mediated by environmental components that facilitate the dynamic coupling of the networks. The dynamic feedback-driven intercommunication of the networks is accomplished via information transfer between the two CDNs effected by hairpin fuel strands in the environment of the system, leading to the coupling of the photochemical and dark-operating modules.

While I am impressed by the complexity of the system, I would still like the authors to comment on three important points in the discussion. First, the dynamic response is not driven by light (the environmental condition), but by the addition of an external compound, which is different from what happens in plants and/or living organisms.
Second, in their system, the first and second network are not coupled with each other, which is of course different to biological systems, where networks dynamically rearrange and reform to perform new functions. In other words: in photosynthesis, when the night sets in, less redox power is provided, which leads ultimately to a shut down of carbon assimilation and triggers the consumption(!) of glucose in the night. The system presented by the authors "only" changes the level of activity upon activation but does not switch and/or revert (in the absence of the signal) completely.
In respect to the latter point, the authors should thirdly also comment on the limitations of their system. The dynamic response of biological systems spans over several orders of magnitude. Here, it is "only" a factor of four, which is achieved by switching from one state to the other state. While this is achieved by an elaborate network, it does still not mimic the full complexity of, which very often allow a complete switch from a very basal activity to full activity.

Re: NCOMMS-21-14359-T Title: Integration of photocatalytic and dark-operating catalytic biomimetic transformations through DNA-based constitutional dynamic networks
The following point-by-point corrections were introduced into the paper.

Reviewer #1
I appreciate the statement of the reviewer that his comments were properly addressed in the revised manuscript.
The reviewer request to "add a remark in the conclusion part that future efforts should be directed to achieve coupling of both CDNs by light irradiation and not only by DNA strands".
Response: Indeed, this is a valid point that was addressed in the conclusion paragraph by the statement: "Beyond advancing "System Chemistry" by introducing artificial dynamic networks mimicking the photosynthetic/plant metabolic processes, important challenges are ahead of us. These include the photochemical coupling of the photosynthetic and metabolic modules and the development of gated and cascaded, dissipative, out-of-equilibrium photosynthetic/metabolic networks." Furthermore, since the second reviewer addressed the same comment by requesting a discussion on the photochemical coupling of the networks in the main text, we added a dedicated section to the main text explaining how the photochemical coupling of the networks can be eventually achieved, page 10, stating: "It should be noted, however, that in contrast to natural photosynthesis, where light triggers the intercommunication of the photosynthetic and metabolic modules, the present study applies "chemical" fuel strands, generated by cleavage of the hairpin structures, as intercommunicating activators. Nonetheless, recent advances in operating dynamic networks demonstrated the light-induced separation of trans-azobenzene stabilized nucleic acid duplexes by photoisomerization of trans-units into the cis-azobenzene state (Angew. Chem. Int. Ed. 2018, 57, 8105-8109). Such photochemical transformation could mimic the natural system by providing photogenerated fuels that guide the intercommunication of the networks."

Reviewer #2
I appreciate the comment of the reviewer that "the manuscript has improved a lot and the central motivation of the study was presented".
The reviewer had three very valid comments that are addressed in the corrected paper.

Comment 1: The dynamic response is not driven by light (the environmental condition), but by the addition of an external compound, which is different from what happens in plants and/or living organisms.
Response: Indeed, the reviewer is correct that in nature the coupling between the photosynthetic module and the metabolic module is driven by light rather than using chemical inputs (nucleic acid fuel strands). This issue was addressed in the discussion section, page 10, by adding a dedicated paragraph explaining how photochemical coupling between the modules can be achieved. The added paragraph states: "It should be noted, however, that in contrast to natural photosynthesis, where light triggers the intercommunication of the photosynthetic and metabolic modules, the present study applies "chemical" fuel strands, generated by cleavage of the hairpin structures, as intercommunicating activators. Nonetheless, recent advances in operating dynamic networks demonstrated the light-induced separation of trans-azobenzene stabilized nucleic acid duplexes by photoisomerization of trans-units into the cis-azobenzene state. Such photochemical transformation could mimic the natural system by providing photogenerated fuels that guide the intercommunication of the networks."

Comment 2:
In photosynthesis, when the night sets in, less redox power is provided, which leads ultimately to a shut down of carbon assimilation and triggers the consumption(!) of glucose in the night. The system presented by the authors "only" changes the level of activity upon activation but does not switch and/or revert (in the absence of the signal) completely.

Response:
The comment that the present networks do not follow the natural principle where the blockage of the photosynthetic module stops the carbon assimilation cycle is certainly correct, and we appreciate clarification of this point by the reviewer.
This comment was explicitly explained and emphasized in the discussion section page 10 and page 11. In fact, we provide a future potential direction to resolve the issue. This comment was addressed and explained as follows: "The blockage of the photosynthetic module in nature (dark conditions) inhibits autonomously the carbon assimilation process, a feature missing in the present intercommunicated artificial networks, due to the irreversible formation of the cleaved hairpin fuel strands. This limitation could be resolved, however, by applying photoisomerizable "activator" strands, such as the cis-azobenzene trigger. The intrinsic "dark" photoisomerization of the cis-azobenzene strand to the trans-state, could then provide an autonomous path to inhibit the metabolic cycle, in analogy to inhibition of the carbon assimilation process under dark conditions of the photosynthetic apparatus.

Comment 3:
The authors should also comment on the limitations of their system. The dynamic response of biological systems spans over several orders of magnitude. Here, it is "only" a factor of four, which is achieved by switching from one state to the other state. While this is achieved by an elaborate network, it does still not mimic the full complexity of, which very often allow a complete switch from a very basal activity to full activity.
Response: Indeed, we agree that the present system reveals limited switching efficiency in contrast to the substantially enhanced switching response of the biological system. We address this comment on page 11, explaining the origin for the present limitation of the dynamic networks and provide a future path to overcome this limitation by dissipative, out-of-equilibrium networks. Appropriate references are included (J. Am. Chem. Soc. 2021, 143, 5071-5079;J. Am. Chem. Soc. 2020, 142, 17480-17488). The comment was addressed as follows: "A further drawback of the present light/dark intercommunicating networks originates from the operation of the coupled CDNs under thermodynamic control. This leads to a permanent background outputs of the CDNs, and to a limited dynamic range of the switching performance of the coupled networks, far lower than in nature. A strategy to overcome this limitation could involve the activation of the photosynthetic/metabolic networks under out-of-equilibrium, dissipative conditions. Indeed, the operation of dissipative CDNs and of gated networks were recently reported (J. Am. Chem. Soc. 2021, 143, 5071-5079;J. Am. Chem. Soc. 2020, 142, 17480-17488), thus providing a path to follow."