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An experimental study of the putative mechanism of a
synthetic autonomous rotary DNA nanomotor
K. E. Dunn, M. C. Leake, A. J. M. Wollman, M. A. Trefzer, S. Johnson and A. M. Tyrrell
Article citation details
R. Soc. open sci. 4: 160767.
http://dx.doi.org/10.1098/rsos.160767
Review timeline
Original submission:
Revised submission:
Final acceptance:
30 September 2016
15 February 2017
23 February 2017
Note: Reports are unedited and appear as
submitted by the referee. The review history
appears in chronological order.
Review History
RSOS-160767.R0 (Original submission)
Review form: Reviewer 1
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
© 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use,
provided the original author and source are credited
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2
Recommendation?
Accept as is
Comments to the Author(s)
Dunn et al. demonstrated the construction of an autonomous rotary DNA nanomotor, which
holds interest for the field of DNA nanotechnology and beyond. As a prototype, though without
a direct observation of rotation, I think the design is solid, and characterizations indirectly
confirmed the strand displacement modulated DNA nanomotor rotation. Therefore I recommend
the publication of this work without further revision.
Review form: Reviewer 2 (Veikko Linko)
Is the manuscript scientifically sound in its present form?
No
Are the interpretations and conclusions justified by the results?
No
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
Recommendation?
Major revision is needed (please make suggestions in comments)
Comments to the Author(s)
The authors provide interesting aspects in building a rotary apparatus from DNA and using
strand displacement reactions to operate it. However, I have several concerns regarding the
work, please see below:
- The authors should characterize their square structures by using AFM or TEM. The authors
mention in the text that they cannot discriminate the unfolded and folded squares with agarose
gel electrophoresis. Therefore, it is even more crucial to see the actual shape of the structures and
the motor (squares connected).
- The gel electrophoresis image in Fig. 4d is extremely hard to interpret and the gel bands do not
look convincing. Even with the analysis in the Supporting information the image is not easily
interpreted.
- I suggest that the authors add schematic and simple figures for describing each step in the
operating procedure (for the gels and for the QCM-D measurements) rather than just acronyms
that are hard to interpret.
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3
- I'm not sure if it is a good strategy to speculate with the further measurements at this level in the
paper without performing them (sections 3d-f). Moreover, I suggest to remove Fig. 5 since that
kind of motor has not been demonstrated in the paper. However, if the authors decide to keep the
speculation of how to realize the motor by using DNA origamis (or other nanostructures), I
would suggest that the authors cite some recent reviews that summarize the currently available
methods to build with DNA at the nanoscale.
Review form: Reviewer 3
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
Recommendation?
Accept with minor revision (please list in comments)
Comments to the Author(s)
Dunn et al describe design and characterization of a DNA nanomotor. This is an interesting study
worthy of publication.
However, there is one main concern. While the title states that the manuscript describes a rotary
motor, the authors do not demonstrate that their construct is indeed rotating (as they themselves
admit, see section d).
Their results, either QMC-D and electrophoresis (not definitive), only show that the system is
capable to go from state 1 (wheel that contains the tape hybridized) to a final state (the unfolded
tape from the wheel staples that form a ds tape), but do not demonstrate that the interconversion
process is a stepwise, rotary process.
Moreover, there are features of the construct that could enable alternative pathways other than
the rotary pathway they describe. For instance, as the tapes (T and T*) are complementary, the
corresponding staples (that make up the wheel) are complementary too (for example St74 and
Str47). Therefore, after strand displacement, when the first bit of both of the staples is released
from T (and T*), these ssDNA segments could also act as a toehold on an alternative strand
displacement route e.g. opening up the wheel, etc. This would, in theory, inhibit the rotary
pathway proposed.
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4
I believe these points need addressing prior to publication either by being upfront about the lack
of proof of rotation, e.g. changing the title to “ A… study of a putative ….” or by additional
experiments.
Decision letter (RSOS-160767)
15-Dec-2016
Dear Dr Dunn,
Thank you for submitting your paper ("An experimental study of a synthetic autonomous rotary
DNA nanomotor driven by strand displacement") for our consideration. Following some delays
due to difficulties in securing reviewers' reports, for which I apologise, we have now received all
reviewers' comments. Based on their reports we would like to invite you to revise your paper in
accordance with the reviewer and Associate Editor suggestions which can be found below. In
essence, the reviewers felt that in its current state the data presented do not support the
conclusions drawn as it was not shown that the motor is rotating. Please either provide
additional experimental data to support your claims or revise the manuscript accordingly.
Furthermore, sections 3d-f are pure speculation and should either be removed or significantly be
condensed. Please note this decision does not guarantee eventual acceptance.
Please submit a copy of your revised paper within six weeks (i.e. by the 26th January 2017). If we
do not hear from you within this time then it will be assumed that the paper has been withdrawn.
In exceptional circumstances, extensions may be possible if agreed with the Editorial Office in
advance. We do not allow multiple rounds of revision so we urge you to make every effort to
fully address all of the comments at this stage. If deemed necessary by the Editors, your
manuscript will be sent back to one or more of the original reviewers for assessment. If the
original reviewers are not available we may invite new reviewers.
To revise your manuscript, log into http://mc.manuscriptcentral.com/rsos and enter your
Author Centre, where you will find your manuscript title listed under "Manuscripts with
Decisions." Under "Actions," click on "Create a Revision." Your manuscript number has been
appended to denote a revision. Revise your manuscript and upload a new version through your
Author Centre.
When submitting your revised manuscript, you must respond to the comments made by the
referees and upload a file "Response to Referees" in "Section 6 - File Upload". Please use this to
document how you have responded to the comments, and the adjustments you have made. In
order to expedite the processing of the revised manuscript, please be as specific as possible in
your response.
In addition to addressing all of the reviewers' and editor's comments please also ensure that your
revised manuscript contains the following sections as appropriate before the reference list:
• Ethics statement (if applicable)
If your study uses humans or animals please include details of the ethical approval received,
including the name of the committee that granted approval. For human studies please also detail
whether informed consent was obtained. For field studies on animals please include details of all
permissions, licences and/or approvals granted to carry out the fieldwork.
• Data accessibility
It is a condition of publication that all supporting data are made available either as
supplementary information or preferably in a suitable permanent repository. The data
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5
accessibility section should state where the article's supporting data can be accessed. This section
should also include details, where possible of where to access other relevant research materials
such as statistical tools, protocols, software etc can be accessed. If the data have been deposited in
an external repository this section should list the database, accession number and link to the DOI
for all data from the article that have been made publicly available. Data sets that have been
deposited in an external repository and have a DOI should also be appropriately cited in the
manuscript and included in the reference list.
If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify
your current submission to dryad, please use the following link:
http://datadryad.org/submit?journalID=RSOS&manu=RSOS-160767
• Competing interests
Please declare any financial or non-financial competing interests, or state that you have no
competing interests.
• Authors’ contributions
All submissions, other than those with a single author, must include an Authors’ Contributions
section which individually lists the specific contribution of each author. The list of Authors
should meet all of the following criteria; 1) substantial contributions to conception and design, or
acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it
critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the
acknowledgements.
We suggest the following format:
AB carried out the molecular lab work, participated in data analysis, carried out sequence
alignments, participated in the design of the study and drafted the manuscript; CD carried out
the statistical analyses; EF collected field data; GH conceived of the study, designed the study,
coordinated the study and helped draft the manuscript. All authors gave final approval for
publication.
• Acknowledgements
Please acknowledge anyone who contributed to the study but did not meet the authorship
criteria.
• Funding statement
Please list the source of funding for each author.
Once again, thank you for submitting your manuscript to Royal Society Open Science and I look
forward to receiving your revision. If you have any questions at all, please do not hesitate to get
in touch.
Yours sincerely,
Alice Power
Royal Society Open Science
on behalf of Katrin Rittinger
Subject Editor, Royal Society Open Science
[email protected]
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6
Comments to Author:
Reviewers' Comments to Author:
Reviewer: 1
Comments to the Author(s)
Dunn et al. demonstrated the construction of an autonomous rotary DNA nanomotor, which
holds interest for the field of DNA nanotechnology and beyond. As a prototype, though without
a direct observation of rotation, I think the design is solid, and characterizations indirectly
confirmed the strand displacement modulated DNA nanomotor rotation. Therefore I recommend
the publication of this work without further revision.
Reviewer: 2
Comments to the Author(s)
The authors provide interesting aspects in building a rotary apparatus from DNA and using
strand displacement reactions to operate it. However, I have several concerns regarding the
work, please see below:
- The authors should characterize their square structures by using AFM or TEM. The authors
mention in the text that they cannot discriminate the unfolded and folded squares with agarose
gel electrophoresis. Therefore, it is even more crucial to see the actual shape of the structures and
the motor (squares connected).
- The gel electrophoresis image in Fig. 4d is extremely hard to interpret and the gel bands do not
look convincing. Even with the analysis in the Supporting information the image is not easily
interpreted.
- I suggest that the authors add schematic and simple figures for describing each step in the
operating procedure (for the gels and for the QCM-D measurements) rather than just acronyms
that are hard to interpret.
- I'm not sure if it is a good strategy to speculate with the further measurements at this level in the
paper without performing them (sections 3d-f). Moreover, I suggest to remove Fig. 5 since that
kind of motor has not been demonstrated in the paper. However, if the authors decide to keep the
speculation of how to realize the motor by using DNA origamis (or other nanostructures), I
would suggest that the authors cite some recent reviews that summarize the currently available
methods to build with DNA at the nanoscale.
Reviewer: 3
Comments to the Author(s)
Dunn et al describe design and characterization of a DNA nanomotor. This is an interesting study
worthy of publication.
However, there is one main concern. While the title states that the manuscript describes a rotary
motor, the authors do not demonstrate that their construct is indeed rotating (as they themselves
admit, see section d).
Their results, either QMC-D and electrophoresis (not definitive), only show that the system is
capable to go from state 1 (wheel that contains the tape hybridized) to a final state (the unfolded
tape from the wheel staples that form a ds tape), but do not demonstrate that the interconversion
process is a stepwise, rotary process.
Moreover, there are features of the construct that could enable alternative pathways other than
the rotary pathway they describe. For instance, as the tapes (T and T*) are complementary, the
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
7
corresponding staples (that make up the wheel) are complementary too (for example St74 and
Str47). Therefore, after strand displacement, when the first bit of both of the staples is released
from T (and T*), these ssDNA segments could also act as a toehold on an alternative strand
displacement route e.g. opening up the wheel, etc. This would, in theory, inhibit the rotary
pathway proposed.
I believe these points need addressing prior to publication either by being upfront about the lack
of proof of rotation, e.g. changing the title to “ A… study of a putative ….” or by additional
experiments.
Author's Response to Decision Letter for (RSOS-160767)
See Appendix A.
RSOS-160767.R1 (Revision)
Review form: Reviewer 2
Is the manuscript scientifically sound in its present form?
Yes
Are the interpretations and conclusions justified by the results?
Yes
Is the language acceptable?
Yes
Is it clear how to access all supporting data?
Yes
Do you have any ethical concerns with this paper?
No
Have you any concerns about statistical analyses in this paper?
No
Recommendation?
Accept as is
Comments to the Author(s)
Dunn et al. have put a significant effort in improving the manuscript. The paper is more concise
and compact after the speculative parts have been removed. Moreover, new gel electrophoresis
data and explanations with additional AFM images make the claims more convincing. In short,
the authors have addressed all my concerns. Therefore, I recommend publication of this
manuscript.
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8
Decision letter (RSOS-160767.R1)
23-Feb-2017
Dear Dr Dunn,
Thank you for submitting a revised version of your manuscript entitled "An experimental study
of the putative mechanism of a synthetic autonomous rotary DNA nanomotor". I am pleased to
inform you that it is now accepted for publication in Royal Society Open Science.
You can expect to receive a proof of your article in the near future. Please contact the editorial
office ([email protected] and [email protected]) to let us know if
you are likely to be away from e-mail contact. Due to rapid publication and an extremely tight
schedule, if comments are not received, your paper may experience a delay in publication.
Royal Society Open Science operates under a continuous publication model
(http://bit.ly/cpFAQ). Your article will be published straight into the next open issue and this
will be the final version of the paper. As such, it can be cited immediately by other researchers.
As the issue version of your paper will be the only version to be published I would advise you to
check your proofs thoroughly as changes cannot be made once the paper is published.
In order to raise the profile of your paper once it is published, we can send through a PDF of your
paper to selected colleagues. If you wish to take advantage of this, please reply to this email with
the name and email addresses of up to 10 people who you feel would wish to read your article.
On behalf of the Editors of Royal Society Open Science, we look forward to your continued
contributions to the Journal.
Kind regards,
Alice Power
Royal Society Open Science
[email protected]
http://rsos.royalsocietypublishing.org/
Reviewer(s)' Comments to Author:
Reviewer: 2
Comments to the Author(s)
Dunn et al. have put a significant effort in improving the manuscript. The paper is more concise
and compact after the speculative parts have been removed. Moreover, new gel electrophoresis
data and explanations with additional AFM images make the claims more convincing. In short,
the authors have addressed all my concerns. Therefore, I recommend publication of this
manuscript.
Appendix A
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Department of Electronics
University of York
Heslington, York
YO10 5DD
UK
Prof. Katrin Rittinger
Royal Society Open Science
The Royal Society
6-9 Carlton House Terrace
London, SW1Y 5AG
14th February 2017
Dear Prof. Rittinger,
Re: Manuscript, ID RSOS-160767
An experimental study of the putative mechanism of a synthetic autonomous rotary
DNA nanomotor (new title), Dunn et al.
Many thanks for your email of 15th December 2016, providing the reviewers’ reports on the
above manuscript and inviting resubmission of a revised manuscript. We thank the reviewers
for their comments and we are grateful for them for taking the time to examine the paper.
Reviewer 1 recommended publication without revision, while reviewers 2 & 3 indicated that
the work is interesting but expressed some reservations. The paper has now been reworked to
address their concerns, in accordance with your request to provide additional data or revise
the manuscript. Extra experiments have been carried out, figures have been modified, the text
has been changed, the conclusions have been revisited and additional references have been
cited.
A detailed point-by-point response to the reviewers’ reports is provided on the following
pages, from which it will be seen that all comments have been addressed in detail.
I trust that the revised manuscript will meet with your approval and look forward to hearing
from you shortly.
Yours sincerely,
Dr Katherine Dunn
Manuscript ID RSOS-160767
An experimental
of the putative mechanism
of a18,synthetic
autonomous rotary
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from http://rsos.royalsocietypublishing.org/
on June
2017
DNA nanomotor (new title), Dunn et al.
POINT-BY-POINT RESPONSE TO THE REVIEWERS’ REPORTS
COMMENT
RESPONSE
Editor
Please either provide additional
experimental data to support your
claims or revise the manuscript
accordingly. Furthermore,
sections 3d-f are pure speculation
and should either be removed or
significantly be condensed.
As described below, additional experimental data has
been provided, and the manuscript has been revised. The
claims have been modified, such that they are now fully
supported by the experimental data. The limitations of the
present study are clearly acknowledged.
Sections 3d-f have been significantly condensed in
accordance with the recommendations made by reviewer
2; the remaining material is required to address comments
made by reviewer 3.
Reviewer 1
Dunn et al. demonstrated the
No action required.
construction of an autonomous
rotary DNA nanomotor, which
holds interest for the field of
DNA nanotechnology and
beyond. As a prototype, though
without a direct observation of
rotation, I think the design is
solid, and characterizations
indirectly confirmed the strand
displacement modulated DNA
nanomotor rotation. Therefore I
recommend the publication of this
work without further revision.
Reviewer 2
The authors provide interesting
aspects in building a rotary
apparatus from DNA and using
strand displacement reactions to
operate it. However, I have
several concerns regarding the
work, please see below:
The authors should characterize
In accordance with the reviewer’s suggestion, AFM
their square Downloaded
structuresfrom
by http://rsos.royalsocietypublishing.org/
using
imaging of the squares
and2017
motor has now been
on June 18,
AFM or TEM. The authors
conducted by an experienced AFM operator (KD) with a
mention in the text that they
recently-installed state-of-the-art microscope. The
cannot discriminate the unfolded
structures proved to be extraordinarily difficult to image
and folded squares with agarose
because the squares were extremely small (which could
gel electrophoresis. Therefore, it
also cause difficulties for TEM imaging). When deposited
is even more crucial to see the
on mica, the area in contact with the surface was expected
actual shape of the structures and to be equivalent to only 100 base pairs for a single square,
the motor (squares connected).
and this led to poor substrate adhesion, even in the
presence of nickel. Consequently, most of the images
were unclear, but it was possible to discern a small
number of objects that were approximately the right size
and shape. The structures were not well-resolved,
presumably because they were bound poorly to the mica.
As a result, the data has not been included in the
manuscript itself, but is presented below.
The reviewer highlighted the finding that it was not
possible to discriminate between unfolded and folded
squares using agarose gel electrophoresis. To allay any
concerns in this respect, polyacrylamide gel
electrophoresis has now been used to characterize the
structures. For small structures, polyacrylamide gels offer
better resolution than agarose gels, and the results
obtained did show a clear difference between the bands
corresponding to unfolded and folded structures, as
expected. This gel is included in Figure 4 and discussed
further in the context of the next point.
The gel electrophoresis image in
Fig. 4d is extremely hard to
interpret and the gel bands do not
look convincing. Even with the
analysis in the Supporting
information the image is not
easily interpreted.
To supplement the new characterization of the structures,
the Supplementary Information now contains the results
of simulations performed using the online tool NUPACK.
This data demonstrates that each domain should primarily
bind to its intended target, offering further support for the
claim that the structures fold well.
The agarose gel shown in Fig 4(d) has been removed. The
corresponding analysis and discussion have been deleted.
Polyacrylamide gel electrophoresis was performed, and
this gave rise to a gel with more convincing bands. This
forms the basis of a replacement Fig. 4(d), which is
discussed in the text. The following note has also been
added to the paper: 'However, gel electrophoresis is not
conclusive and provided information only on static
structures. In contrast, the subsequent QCM-D
experiments allowed structural changes to be measured
in real time.' Full details of the polyacrylamide gel
samples are provided in the Supplementary Information.
I suggest that the authors add
Additional arrows have been added to the triangle
schematic and
simplefrom
figures
for
schematics in Fig.
for18,clarity.
Simple schematic
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http://rsos.royalsocietypublishing.org/
on 3,
June
2017
describing each step in the
diagrams have been added to Fig 4c-e, to make it easier to
operating procedure (for the gels
interpret the gels and QCM-D measurements. A reference
and for the QCM-D
to the sketches has been inserted in the caption.
measurements) rather than just
acronyms that are hard to
interpret.
I'm not sure if it is a good strategy The speculation about further measurements has been cut
to speculate with the further
dramatically. Sections 3d-e have been shortened
measurements at this level in the
significantly and combined, such that further experiments
paper without performing them
and design enhancements are now described in one
(sections 3d-f). Moreover, I
section. Section 3f (applications) has been reduced to a
suggest to remove Fig. 5 since
single sentence, now placed at the very end of the
that kind of motor has not been
conclusions, and the corresponding sentence in the
demonstrated in the paper.
abstract has been shortened.
However, if the authors decide to
keep the speculation of how to
The revised paper briefly mentions the possibility that the
realize the motor by using DNA
motor could be realized using DNA origami or other
origamis (or other
nanostructures, but Figure 5 has been removed in
nanostructures), I would suggest
accordance with the reviewer's suggestion. The following
that the authors cite some recent
additional papers have been cited, with reference to the
reviews that summarize the
currently available methods to build with DNA at the
currently available methods to
nanoscale:
build with DNA at the nanoscale.  Rothemund, 'Folding DNA to create nanoscale shapes
and patterns', Nature (2006)
 Castro et al., 'A primer to scaffolded DNA origami',
Nature Methods (2011)
 Chandrasekaran, 'DNA origami and biotechnology
applications: a perspective', J. Chem. Technol.
Biotechnol. (2016)
 Lee Tin Wah et al., 'Observing and controlling the
folding pathway of DNA origami at the nanoscale',
ACS Nano (2016)
 Majikes et al. 'Competitive annealing of multiple
DNA origami: formation of chimeric origami', New J.
Phys. (2016)
 Marras et al. 'Directing folding pathways for multicomponent DNA origami nanostructures with
complex topology', New J. Phys. (2016)
 Han et al., 'DNA Gridiron nanostructures based on
four-arm junctions', Science (2013)
 Ke et al., 'Three-dimensional structures self-assembled
from DNA bricks', Science (2012)
 Benson et al., 'DNA rendering of polyhedral meshes at
the nanoscale', Nature (2015)
 Veneziano et al., 'Designer nanoscale DNA
assemblies programmed from the top down', Science
(2016)
Reviewer 3
Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
Dunn et al describe design and
characterization of a DNA
nanomotor. This is an interesting
study worthy of publication.
However, there is one main
concern. While the title states that
the manuscript describes a rotary
motor, the authors do not
demonstrate that their construct is
indeed rotating (as they
themselves admit, see section d).
Their results, either QCM-D and
electrophoresis (not definitive),
only show that the system is
capable to go from state 1 (wheel
that contains the tape hybridized)
to a final state (the unfolded tape
from the wheel staples that form a
ds tape), but do not demonstrate
that the interconversion process is
a stepwise, rotary process.
Moreover, there are features of
the construct that could enable
alternative pathways other than
the rotary pathway they describe.
For instance, as the tapes (T and
T*) are complementary, the
corresponding staples (that make
up the wheel) are complementary
too (for example St74 and Str47).
Therefore, after strand
displacement, when the first bit of
both of the staples is released
from T (and T*), these ssDNA
segments could also act as a
toehold on an alternative strand
displacement route e.g. opening
up the wheel, etc. This would, in
theory, inhibit the rotary pathway
proposed.
I believe these points need
addressing prior to publication
either by being upfront about the
lack of proof of rotation, e.g.
changing the title to “ A… study
of a putative ….” or by additional
experiments.
As acknowledged in the original paper, it is true that the
present results do not definitively prove that the motor
rotates. For conclusive demonstration that the
interconversion of the initial and final states is a stepwise
rotary process, it would be necessary to perform single
molecule experiments (as mentioned in the text), which
would be extremely challenging. These experiments
would almost certainly necessitate a complete redesign of
the motor, introducing a number of enhancements (as
outlined in the text). This exciting work may be
undertaken in the future but is beyond the scope of the
present manuscript. To address the reviewer's concerns,
the title of the paper has been changed and the text
modified, as described below. The revised wording is
intended to clarify the aims of this study and make it
absolutely clear that this study does not claim to provide
definitive proof of rotation.
The reviewer is correct to point out that alternative
pathways may exist. It is not known how these could
affect the operation of the motor, and this point is now
acknowledged in the paper, as follows: 'Alternative
displacement pathways may exist, and it is not clear what
effect this could have on the operation of the motor. It is
possible that these pathways could be eliminated with an
alternative motor design based on components made
using DNA origami methods, as discussed below.'
The title has been changed as suggested by the reviewer.
The title is now: ‘An experimental study of the putative
mechanism of a synthetic autonomous rotary DNA
nanomotor.’
The text has also been modified. For instance, the abstract
now states:
'This paper describes an experimental study of the
putative mechanism of a rotary DNA nanomotor, which
is based on strand
the phenomenon that
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on displacement,
June 18, 2017
powers many synthetic linear DNA motors. Unlike other
examples of rotary DNA machines, the device described
here is designed to be capable of autonomous operation
after it is triggered. The experimental results are
consistent with operation of the motor as expected, and
future work on an enhanced motor design may allow
rotation to be observed at the single molecule level.'
The last paragraph in the ‘Background’ now states:
This paper describes the experimental study of the
putative mechanism of an autonomous rotary DNA
motor, through a series of experiments in which the
underlying principles were tested and a prototype of the
motor was examined. The motor is designed to be driven
by sequential strand displacement reactions, and the
concept is explained in the Methods section, in which the
experimental procedures are also described. […] The
experimental results were consistent with the motor
operating as intended, but the methods used here did not
allow rotation to be seen directly. The aims of the
present study were (a) to validate the hypothesis that
sequential strand displacement reactions could catalyse
significant structural rearrangement in a folded DNA
construct on a surface and (b) to construct a rotary
motor prototype based on this idea, confirming that it
undergoes a structural transition as a result of strand
displacement. Further experiments on an enhanced
motor design may build on this work by imaging
rotation with single-molecule resolution, and this is
discussed towards the end of the paper.’
The concluding section now begins: ‘This paper has
described an experimental study of the putative
mechanism of a prototype synthetic rotary motor made
from DNA. The motor was designed to be driven by
strand displacement and to be capable of autonomous
operation after the brake was released.’
The record in the Dryad data repository has been updated
to reflect the changes. As indicated, additional
experiments may form the basis of follow-up projects.
The manuscript contains the following sections before the reference list, as requested:
 Ethics statement (confirming that the work did not require ethical approval)
 Data accessibility (stating where data can be accessed; dataset is also cited in references)
 Competing interests (statement that a patent has been filed for the rotary motor concept)
 Authors’ contributions (listing the specific contributions of each author)
 Funding statement (the source of funding is listed for each author)
No additional acknowledgement section was required for this work.
ADDITIONAL EXPERIMENTS PERFORMED: AFM IMAGING OF SQUARES
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AND MOTOR
Figures (on next two pages)
(a), (c): Sketches of the expected appearance of squares of type B (a) and the assembled
motor, with the squares connected (c). The blue rectangles represent the helices forming the
structure. The rectangles with a thicker outline represent areas where two helices are
superimposed. Diagrams are approximately to scale, assuming the standard parameters for BDNA in solution. Dimensions were calculated in the absence of broadening effects.
(b), (d): AFM images of Square B (b) and assembled motor (d). Each sample was prepared as
for the polyacrylamide gel experiment, and 10L was deposited on freshly cleaved mica that
had been washed with 10L of 50mM NiCl2. The mica was swirled to distribute structures
across the surface and the specimen was left for several minutes for surface adsorption. AFM
imaging was then performed in tapping mode in fluid using a Bruker Bioscope Resolve, with
Bruker SNL probes (cantilever C). The imaging buffer is given in the figures. Images were
processed using the program Gwyddion, employing plane levelling, row alignment, contrast
adjustment, shifting minimum data value to zero and profile extraction. The line graphs show
the height profile along the lines indicated in the accompanying image.
Discussion of AFM data provided after figures.
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Downloaded from http://rsos.royalsocietypublishing.org/ on June 18, 2017
Discussion of AFM data
As noted above, the structures were not well-resolved. Furthermore, very few structures were
observed, despite the comparatively high concentration (1-2M) and the presence of nickel,
which usually aids adhesion. The low resolution and the scarcity of structures to image were
both attributable to poor adhesion to the mica, due to the small size of the structures.
However, taking into account the effects of tip broadening, poor adhesion and flexibility
within the structures, the shape and size of the objects seen in the AFM images are consistent
with expectations.
Assuming that a DNA helix is 2nm in diameter, the squares and motor would be expected to
be around 2-5nm high. The line profiles in the figures show that the objects imaged were
between 2 and 4 nm in height, consistent with the prediction.