Sites of high local frustration in DNA origami

The self-assembly of a DNA origami structure, although mostly feasible, represents indeed a rather complex folding problem. Entropy-driven folding and nucleation seeds formation may provide possible solutions; however, until now, a unified view of the energetic factors in play is missing. Here, by analyzing the self-assembly of origami domains with identical structure but different nucleobase composition, in function of variable design and experimental parameters, we identify the role played by sequence-dependent forces at the edges of the structure, where topological constraint is higher. Our data show that the degree of mechanical stress experienced by these regions during initial folding reshapes the energy landscape profile, defining the ratio between two possible global conformations. We thus propose a dynamic model of DNA origami assembly that relies on the capability of the system to escape high structural frustration at nucleation sites, eventually resulting in the emergence of a more favorable but previously hidden state.

1. C an the authors comment on the mechanical stresses/folding trajectories involved when folding a circular scaffold as compared to that of a linear scaffold? 2. Page 11, line 12: This latter was shown …. -> This later was shown…. 3. Figure 2

caption. (d) -> (e)
Reviewer #2: Remarks to the Author: By bringing the possibility to program the assembly of DNA brick into virtually any kind of desired functional nanostructures, the so-called « DNA origami » is one of the most important selfassembly methods invented in the 21st century, with a particularly mighty impact in nanoscience. Because this method is exceptionally robust, high-yield and practically flawless, it has been adopted by a huge number of scientists. Surprisingly, very little is known about the folding pathways of DNA origamis, how to guide it and how to re-orientate it so that non-canonical shapes could be obtained. This is precisely these three challenges that are addressed by this impressive manuscript. By systematic and smart changes of edge staple compositions, the author reliably establish the probability reach different origami isomers as a function of the mechanical stress experienced during the initial folding steps. Very interestingly, they can further build an energy landscape of DNA origami, which is shown to be adaptive and eventually completely reorientate the folding toward structures that are otherwise unfavored. This not only dramatically increase our understanding of DNA origami folding process but provides also keys to redirect the folding into non-canonical shapes. The experimental design is particularly smart. The conclusions are very solid and based on a huge number of reliable data. The data analysis is also particularly complete. Overall, although very dense, the manuscript is well written and pleasant to read. For all of these reasons, I recommend the publication of this manuscript in Nature C ommunications. I just have a few suggestions of minor modification, and they all concern the presentation. 1) Although I find the manuscript rich and clear (introduction, results, conclusions, figures), I feel that the abstract does not convey well the manuscript content, by being a bit too general and too specific at the same time. It uses some technical terms (iso I and iso II isomers), which might be difficult to understand for a non-expert readers. I would also suggest to explain better the experimental approach through the use of edge staples to direct the folding.
2) In figures 2 and 3, I would suggest to plot the distribution of isomers obtained for each edge composition rather than the opposite, which for me would be more straightforward to analyze. This is of course just a question of presentation and I let the authors decide what presentation they estimate to be the most relevant.

Sites of high local frustration in DNA origami
Richard Kosinski, Ann Mukhortava, Wolfgang Pfeifer, Andrea Candelli, Philipp Rauch, and Barbara Saccà We are extremely glad for the enthusiastic comments received from the Reviewers and the positive support of the Editor. We feel that our work has been sincerely appreciated and, with it, we hope to have contributed to advance our understanding of DNA origami self-assembly: a robust and widely-used method, still holding intriguing aspects.
Below are our point-by-point responses to the Reviewers´ comments.

Reviewer #1 (Remarks to the Author):
This exciting study merges the topological and sequence dependent views to study the folding of monolayer DNA origami and provides quantitative data to trace the full energy landscape of folding. To understand the topological constraints in folding of DNA origami, the authors designed reconfigurable DNA origami structures that has two energy minima states; canonical (iso I) state where antiparallel stretches of scaffold strand are kept in place by staple crossovers and iso II isomer state where antiparallel stretches of staples are joined by scaffold crossovers. The authors studied folding in the presence and absence of different types of edges staples to induce scaffold inversions adding topologically stressed sites. Additionally, to understand the role of sequence depending in folding, the authors studied folding of three quasi-independent domains of identical shape but distinct sequence content. The authors propose a dynamic model of DNA origami folding based on their observations that the formation of topologically stressed sites reshapes the energy landscape of the assembly process and dictates the populations of the two isomers. The authors identify the degree of mechanical stresses and their role in early stages of nucleation as key factors influencing the outcome of DNA origami folding. The general subject of the study fits the journal scope quite well and will be useful to the broad scientific community. I recommend this article to be published in Nature Communications, after addressing the following minor comments: We sincerely thank this reviewer for the positive evaluation of our work.
1. Can the authors comment on the mechanical stresses/folding trajectories involved when folding a circular scaffold as compared to that of a linear scaffold? We thank this reviewer for raising up this very good point. At the beginning of our study, we have indeed thought that the circularity of the scaffold could have been one source of unsustainable topological stress leading to formation of an alternative and mechanically more stable form. Accordingly, we reasoned that a linear scaffold should permit to release the excessive tension and enable formation of a higher amount of canonical structures. To prove this hypothesis, we introduced a nick into the circular scaffold at one selected site located within the core of domain A and performed the self-assembly of the full structure using the so-obtained linear scaffold and edge staples type 0. Our results are presented in the new Supplementary Figure 46. A description of the procedure used for the enzymatic restriction and purification of the linear scaffold is reported in the Supplementary Materials and Methods section. In brief, we did not observe a significant increase in the fraction of canonical species, disproving our initial hypothesis, i.e. meaning that the circularization of the scaffold is not the main reason for the observed conformational change. In view of our further studies, we deduce that this is due to introduction of the nick into a region that is distant from the nucleation strands at the edges and has a lower thermal stability, thus affecting the fate of the assembly only in minimal part. The situation might be different when the nick is introduced at one of the scaffold turns, particularly if involved in the initiation of the assembly process. In this case, we presume that the conditions are met to release the mechanical stress applied without the need for isomerization of the entire domain.
2. Page 11, line 12: This latter was shown …. -> This later was shown…. Corrected into "This was shown to be …".

Reviewer #2 (Remarks to the Author):
By bringing the possibility to program the assembly of DNA brick into virtually any kind of desired functional nanostructures, the so-called « DNA origami » is one of the most important self-assembly methods invented in the 21st century, with a particularly mighty impact in nanoscience. Because this method is exceptionally robust, high-yield and practically flawless, it has been adopted by a huge number of scientists. Surprisingly, very little is known about the folding pathways of DNA origamis, how to guide it and how to re-orientate it so that noncanonical shapes could be obtained. This is precisely these three challenges that are addressed by this impressive manuscript. By systematic and smart changes of edge staple compositions, the authors reliably establish the probability to reach different origami isomers as a function of the mechanical stress experienced during the initial folding steps. Very interestingly, they can further build an energy landscape of DNA origami, which is shown to be adaptive and eventually completely reorientate the folding toward structures that are otherwise unfavored. This not only dramatically increases our understanding of DNA origami folding process but provides also keys to redirect the folding into non-canonical shapes. The experimental design is particularly smart. The conclusions are very solid and based on a huge number of reliable data. The data analysis is also particularly complete. Overall, although very dense, the manuscript is well written and pleasant to read. For all of these reasons, I recommend the publication of this manuscript in Nature Communications. I just have a few suggestions of minor modification, and they all concern the presentation. We are very grateful to this reviewer for the sincere appreciation of our work. 1) Although I find the manuscript rich and clear (introduction, results, conclusions, figures), I feel that the abstract does not convey well the manuscript content, by being a bit too general and too specific at the same time. It uses some technical terms (iso I and iso II isomers), which might be difficult to understand for non-expert readers. I would also suggest to explain better the experimental approach through the use of edge staples to direct the folding.