Peer review of: ATP-powered molecular recognition to engineer transient multivalency and self-sorting 4D hierar (2020)

Editorial Note: This manuscript has been previously reviewed at another journal that is not operating 
a transparent peer review scheme. This document only contains reviewer comments and rebuttal  
letters for versions considered at Nature Communications. 
 

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
This reviewer is not confused; it is the authors that, by using imprecise terminology, send out a 
confusing message to the community. They fail to recognize the difference between different 
classes of fuel-driven systems. The system reported here relies on two subsequent covalent 
reactions (ATP-fuelled DNA-ligation followed by DNA-restriction: A+ATP→B+ADP→A+P, with the 
fortunate case that the final product is the same as the starting material (A). By continuously 
adding ATP the system can be maintained in a dynamic steady state with the intermediate 
compound B populated. 
 
This is different from the examples 1-4 provided by the authors. In these examples, a chemical 
fuel is used to activate a building block altering its self-assembly properties. As a consequence, in 
all these systems two different energy dissipation pathways are present: building blocks can be 
deactivated in the monomeric state or in the assembled state. This is of importance, because the 
relative contributions of these pathways actually determines how chemical energy is exploited by 
the system. The differences have recently been discussed in a series of papers: 
 
Ragazzon, Prins Nat Nanotechnol. 2018 Oct;13(10):882-889 
Astumian Nat.Communications. 2019 10:3837 
Penocchio, Rao, Esposito Nat.Communications. 2019 10:3865 
 
Confusion arises because in this research area the same terminology is used to describe all 
systems without distinction. This is indeed confirmed by the discussion in the peer-review file of 
another paper in this area cited by the authors in their rebuttal letter (I was not that reviewer). 
Considering that the corresponding author is a main contributor in this area, it may be expected 
that he can distinguish the different scenario’s. 
 
Although I see why the authors have used the term out-of-equilibrium, I would suggest not to use 
it. Formally it is true that any ongoing chemical reaction (or sequence) is not at equilibrium, but it 
is not common practice in chemistry to describe chemical reactivity in these terms. What the 
authors have developed here is a cleverly designed way to maintain the system always moving 
towards the thermodynamic end state without ever reaching it. I think the terms dynamic kinetic 
steady state and transientness are very appropriate. I would further suggest to disconnect the 
word dissipative from structure (as in ‘ATP-dissipative structures page 5’ or ‘..to engineer 
dissipative structures’ in the caption of Figure 1). The only structures in the system able to 
dissipate energy are the two enzymes. 
 
Apart from this discussion on the used terminology, I think the work is of a high scientific level and 
offers a new perspective on DNA-nanotechnology. My previous comment on the incremental 
nature referred to the fact that the methodology of creating dynamic steady states had been 
published before. Here, the authors optimize that approach and exploit it. The obtained results are 
suitable for publication in Nature Communications. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
In this work, the authors extemd their previously introduced concept to generate out-of- 
equilibrium dynamic steady states in DNA systems by combining the action of a ligase and a 
restriction endonuclease. In contrast to earlier work, the use of a type II-S restriction 
endonuclease allows to more freely choose the sequence involved (the same idea has been 
previously used in DNA computing and is the foundation of the Golden Gate assembly method). 
With this new capability, they demonstrate the transient formation of DNA polymers, the transient 
assembly of colloidal particles, surface polymerization and also “self sorting”. 
 
This (new) reviewer thinks that after the revisions following the first round of reviewing the paper 
is generally acceptable for publication in Nat. Commun., but wishes to add a few notes. 
 
As similar ideas have been around for a long time, this reviewer would disagree that the approach 
taken here is so radically new to justify statements like “break with these concepts and open a 
new avenue to 4D colloidal assemblies via our transient modular DNA toolbox and using ATP as 
molecular fuel.” (and others) 
 
- for instance, the author emphasizes the difference between the use of ATP as fuel and previous 
work using DNA/RNA polymerization. Still the systems are based on the same idea: one process 
“builds up” a structure, and a competing process degrades it. In fact, all the oscillating systems 
shown before can also show “transient behavior”, when they are not driven in the right parameter 
range. It typically requires considerable fine-tuning to generate oscillations, while it is relatively 
easy to see the transient behavior also reported here – and this is done in part via the ratio of the 
enzymes added … 
Next to the work cited here (Ref. 38), the authors may also like to look at: E. Franco et al., PNAS 
108, E784–93 (2011); J. Kim, Nucleic Acids Res. 42, 6078–6089 (2014). 
The paper by Dehne et al. cited in 39 also shows transient colloid assembly – and the difference to 
the present work (of course, the authors emphasize ATP consumption etc.) is not that large … 
 
- The authors may be interested in the discussion on “fueling” computational processes *without 
ATP* using Type II-S enzymes in Y. Benenson, et al. PNAS, 100, 2191–2196 (2003). 
 
- it is not clear to this reviewer why the authors use the statement “repurpose advanced Golden 
Gate cloning”. Why the word “advanced”? It is not necessary to inflate statements all the time. 
 
- the use of the word “tile” in this manuscript (here it is just used for double strands with sticky 
ends and one type of single strands) differs from the typical use of the term in DNA 
nanotechnology and may be confusing. Also “single-stranded tiles” such as those developed by 
Peng Yin’s group are different as they are characterized by a very specific sequence domain 
structure.