1. Art and Diseño

Nano Research
Nano Res
DOI 10.1007/s12274-015-0724-z
1
Serum Induced Degradation of 3D DNA Box Origami
Observed by High Speed Atomic Force Microscope
Zaixing Jiang1,2,†, Shuai Zhang2,†, Chuanxu Yang2, Jørgen Kjems 2, Yudong Huang1,*, Flemming
Besenbacher 2, Mingdong dong2,*
Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0724-z
http://www.thenanorese arch.com on January 28, 2015
© Tsinghua University Press 2015
Just Accepted
This is a “Just Accepted” manuscript, which has been examined by the peer-review process and has been
accepted for publication. A “Just Accepted” manuscript is published online shortly after its acceptance,
which is prior to technical editing and formatting and author proofing. Tsinghua University Press (TUP)
provides “Just Accepted” as an optional and free service which allows authors to make their results available
to the research community as soon as possible after acceptance. After a manuscript has been technically
edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP
article. Please note that technical editing may introduce minor changes to the manuscript text and/or
graphics which may affect the content, and all legal disclaimers that apply to the journal pertain. In no event
shall TUP be held responsible for errors or consequences arising from the use of any information contained
in these “Just Accepted” manuscripts. To cite this manuscript please use its Digital Object Identifier (DOI®),
which is identical for all formats of publication.
TABLE OF CONTENTS (TOC)
Serum
Induced
Insert your TOC graphics here.
Degradation of 3D
DNA Box Origami
Observed
by
High
Speed Atomic Force
Microscope
Zaixing Jianga,b, Shuai
b,
Zhang
Chuanxu
Yang b, Jørgen Kjems
b,
Yudong Huanga, *,
Flemming
The degradation kinetics of 3D DNA box origami in serum using high speed atomic force
b,
Besenbacher
Mingdong dong
a
b ,*
Department
microscope has been demonstrated. Our findings are valuable for the further modifications to
improve the biocompatibility of DNA nanostructures in future applications.
of
Polymer Science and
Technology, School of
Chemical Engineering
and
Technology,
Harbin
Institute
Technology,
150001,
of
Harbin
People’s
Republic of China
b
Interdisciplinary
Nanoscience
Center
(iNANO),
Aarhus
University, DK-8000,
Aarhus C, Denmark
Provide the authors’ webside if possible.
Author 1, webside 1
Author 2, webside 2
Nano Research
DOI (automatically inserted by the publisher)
Review Article/Research Article Please choose one
Serum Induced Degradation of 3D DNA Box Origami
Observed by High Speed Atomic Force Microscope
Zaixing Jiang1,2,†, Shuai Zhang2,†, Chuanxu Yang2, Jørgen Kjems 2, Yudong Huang1,*, Flemming
Besenbacher 2, Mingdong dong2,*
Received: day month year
ABSTRACT
Revised: day month year
3D DNA origami holds tremendous potential to encapsulate and selectively
release therapeutic drugs. Observations of real-time performance of 3D DNA
origami structures in physiological environment will contribute much to its
further applications. Here, we investigate the degradation kinetics of 3D DNA
box origami in serum using high-speed atomic force microscope optimized for
imaging 3D DNA origami in real time. The time resolution allows
characterizing the stages of serum effects on individual 3D DNA box origami
with nanometer resolution. Our results indicate that the whole digest process is
a combination of a rapid collapse phase and a slow degradation phase. The
damages of box origami mainly happen in the collapse phase. Thus, the
structure stability of 3D DNA box origami should be further improved,
especially in the collapse phase, before clinical applications.
Accepted: day month year
(automatically inserted by
the publisher)
© Tsinghua University Press
and Springer-Verlag Berlin
Heidelberg 2014
KEYWORDS
3D DNA box origami,
high-speed AFM, stability,
serum, kinetic
1 Introduction
As being one recently developed high efficient
simplicity to complicity, with high yields and
self-assembly technique, the DNA origami is one
applications, such as serving as nanoscale rulers for
radically
DNA
single molecule imaging [8], templates for the
nanotechnology [1-4]. By folding a long single DNA
nanowire growth [9, 10], aid in the molecular
strand into arbitrary shapes with hundreds of
structure determination [11, 12], and new platforms
synthetic staple strands, DNA origami has been
for genomics applications [13, 14]. 3D DNA box [15],
proved to form 2D and 3D nanostructure, from
which was first designed and synthesized in 2009, is
increased
subgroup
of
accuracy [5-7]. These nanostructures have many
2
Nano Res.
one kind of 3D DNA origami nanostructures with
hollow core. Due to it has the lid, which can be
opened by certain target gene sequence, it is
considered as potential drug carrier in vivo.
However, one of the major barriers towards the
in vivo applications of DNA origami nanostructures,
including 3D DNA box, is the susceptibility of their
strands and structures towards nuclease degradation
in physiological environments. As being one of
nuclease degradation reagents, serum contains a
mixture of nucleases and proteins, such as endo- and
exo-nucleases, which have the degradation effect to
DNA strands. And according to our best knowledge,
serum has been applied as one kind of standard
reagents to test the susceptibility of other synthesized
DNA nanostructures in vivo [16-19]. Hence, serum is
considered as an ideal touchstone to evaluate the
stability of DNA origami in vivo.
Recently, a number of methods have been used
to study the stability of DNA origami, such as
scanning electron microscope (SEM) [20, 21], atomic
force microscope (AFM) [22, 23], transmission
electron microscope (TEM) [24, 25] and agarose gel
[26, 27]. Among them, AFM is the wide applied one;
as it is of capability to evaluate the stability of DNA
origami based on 3D quantitative morphology, and
provide in situ DNA origami response behaviors to
different stimulates [28, 29]. However, the detailed
evidences of the origami structure stabilities, direct
visualization of the degradation process, and
real-time analysis could not be achieved by standard
AFM, because of its slow innate scan speed [30].
Currently, these drawbacks have been overcome
gradually with the development of high-speed
atomic force microscopy (HS-AFM) [31-34]. HS-AFM
had been employed to characterize dynamic process
of some origami related materials [30, 34, 35]. But as
far as we know, the kinetics of 3D DNA origami
structural evolution in response to external stimuli
has not been explored with HS-AFM.
In this study, we intend to apply HS-AFM to
directly in situ observe the degradation process of 3D
DNA box origami in serum [15], which is meaningful
and instructive to realize its final application in drug
delivery. With the technical developments, the
degradation process of 3D DNA box origami has
been directly observed in real-time. The quantitative
degradation kinetics of 3D DNA box origami has
been then studied. And the critical degradation
concentration for 3D DNA box origami structure
damage is also determined. These data are valuable
for future improvement of DNA origami based drug
carrier.
2 Experimental
2.1 The synthesis of the DNA box
The software package, used for the box design,
consists of a sequence editor and an extendable
algorithm toolbox [1]. A program for creating
realistic 3D models has been developed, which
facilitated the design of the 3D edge-to-edge staple
strand
crossovers.
The
software
package
is
distributed as free software (the GNU General
Public License version 3 (GPLv3)) are available at
www.cdna.dk/origami.
The m13mp18 DNA was prepared as described
previously [1, 15]. The assembly reactions were
performed
in
Tris-acetate-EDTA
buffer
with
12.5mM MgAc (TAEM), 1.6nM M13 and fivefold
excess of each oligonucleotide. The samples were
heated to 95℃ and cooled to 20℃ in steps of 0.1℃
every 6 s. Then, the staple strands that fold the box
by bridging the edges are constructed, resulting in a
‘cuboid’ structure of external size 42×36×36 nm 3
(sequence map and more design details can be seen
in reference 1 an 9 published by our group) [1, 15].
Finally, the lid was functionalized with a lock–key
system to control its opening. The designed DNA
structure formed by self-assembly after heated
annealed
the
220
staple
strands
onto
the
single-stranded M13 DNA, resulting in highly
homogenous structures migrating as one distinct
band in native gel electrophoresis. In an assembly
reaction, 59 staple strands were used, connecting
the edges, to form the box shape.
2.2 Prepare serum solution and inject it into the cell
Fetal Bovine Serum (FBS), heat inactivated, was
supplied by Life Technologies company, and used as
it obtained. The FBS was dilluted by the 1×TAE/Mg2+
| www.editorialmanager.com/nare/default.asp
3
Nano Res.
buffer. The FBS was carefully injected into the AFM
mM EDTA pH:8.0, 12.5 mM MgAc 2) was added. All
cell by our homemade injection system. The injection
measurements were performed in tapping mode in
system was composed by syringe (1.0ml), syringe
fluid. The experiment temperature is about 25℃.
pump (Aladdin-1000, Word Precision Instruments
Flow-through fluid exchange was achieved using a
Programmable), rubber tube (Φ1.0mm), needle (4.5#)
dual syringe pump. Height, amplitude and phase
and needle holder. In order to avoid the bubble
signals were recorded for both trace and retrace.
interference, the FBS was full fill the rubber tube and
The
needle. The AFM was fistly begin to work, and after
AnalysisTM, SPIPTM and Cinema 4DTM, using
it had got at least two images, the injection system
standard modification commands applied over the
begin to work. The FBS was injected automatically by
whole sample.
3 Results and discussion
Imaging 3D hollow DNA box in a biological
environment with high local line speed is one of the
most challenging of bio-applications to AFM [36].
The interaction force between the probe and the
sample is critical. If the force is too high, the 3D DNA
box origami are damaged by the probe, confusing its
own response to serum; if it is too low, resolution of
the image is unsatisfactory due to the weak feedback.
More thorny issue is that the interaction force should
be controlled in high speed scanning process. It is
known that the scanning speed of AFM is proportion
to individual resonant frequency. The resonant
frequency fc and the spring constant kc of a
rectangular cantilever with thickness d, width w, and
length L are expressed as [30]:
the injection system. The DNA buffer should be also
precisely added onto the AFM tip and cell, an the
total amout of liquid is about 100μl including the
FBS.
2.3 Survival percent defination
Every data point of survival percent is counted
from more than 50 3D DNA origami box. We define
that the origami box is died when its height reduce to
half of its original height according to the assumptio
that if one of its surface is lost, the loadings in the box
will be leaking; but the loadings will be greatly
released if half of its original height is remained, i.e.
it has been already collapsed. Thus, we define the
above standard for whether the box is died or living.
data
were
processed
using
NanoScope
The height of the box is calculated from software of
SPIPTM.
2.4 High-speed Atomic force microscopy
AFM images were obtained using Dimension
and
FastScan AFM (Bruker, CA) with FastScan-C
cantilevers. The FastScan-C cantilevers utilize a
novel 40 μm long triangular Silicon Nitride
cantilever to achieve a 70-150 kHz resonant
Where E and ρ are Young’s modulus and the
frequency in liquid with only a 0.4-1.2 N/m force
density of the used material, respectively. To attain a
constant. The Silicon tip has an extremely sharp
high resonant frequency and a small spring constant
with 5 nm tip radius, making it ideal for imaging a
simultaneously, cantilevers with small dimensions
wide variety of hard and soft materials. All
must be fabricated. In addition to the advantage in
FastScan cantilevers have less that 3 degrees of
achieving a high imaging rate, small cantilevers have
cantilever bend.
other advantages, such as lower noise density, less
During the experiment, the sample (1 μL) was
affected by thermal noise and high sensitivity to the
adsorbed onto a freshly cleaved mica plate (ϕ12
gradient of the force exerted between the tip and the
mm, Bruker, CA) for 1min at room temperature,
sample. Thus, only small cantilever can balance the
and then 1×TAE/Mg2+ buffer (40 mM Tris pH:8.0, 2
high speed and low force on sample. In this work,
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
4
Nano Res.
the small cantilevers, made of silicon nitride and
disruption of 3D DNA box origami structure caused
coated with gold of about 20 nm thickness, are used.
by AFM tip, the acquisition time will be doubled
They have resonance frequencies in liquid at ~80 kHz
after every ten images. Four images obtained at the
and spring constants between 0.4 and 1.2 nN/nm.
critical point in time are shown in Figure 3a (for the
The higher resonance frequency, the lower mass, and
full series see Supplementary movie 2). It is evident
smaller spring constant enable the imaging speed in
that most of 3D DNA boxes have a similar respond
tapping mode to be increased while keeping the
time to the addition of serum solution. Figure 3b
imaging forces on the 3D DNA box origami small.
shows the survival percent values for the 3D DNA
Figure 1a shows a scanning electron microscopy
box origami as a function of time after the addition of
(SEM) image of the small cantilever used in this
serum. Time of degradation is highly variable, with
research. The inset is a comparison between a
an
conventional cantilever and the small one. Figure 1b
concentration-dependent
shows the thermal spectra of the two types of
executed with lower (0.01 vol% and 0.001 vol%) and
cantilevers. The resonance frequency of the small
higher
cantilever is almost 20 times higher than that of the
corresponding results are shown in Figure 3c. At
conventional cantilever in fluid.
lower serum concentration, no degradation of the 3D
average
(1.0
vol%)
of
62±26s.
experiments
Serum
are
also
serum concentrations. The
Before serum was injected, the morphology of
DNA box origami is observed (for the full series see
3D DNA box was characterized in advance. The
supplementary movie 3 and 4). The slope of the two
sample was prepared and adsorbed onto a freshly
plots almost has no change, i.e. the degradation
cleaved mica substrate. After adding the observation
speed approaching zero (see Figure S1 and S2 in the
buffer
Electronic
containing
Mg ,
2+
the
high-speed
AFM
Supplementary
Material
(ESM)).
experiment was started with a larger scan area to
Conversely, at 1 vol% serum addition, an abrupt
investigate morphology of 3D DNA box origami.
change is happened in the beginning, i.e. all of 3D
These results are shown in Figure 2 (for the full series
DNA boxes are destroyed immediately after the
see Supplementary movie 1). As seen, there remains
serum addition (for the full series see Supplementary
lots of DNA sheets on mica surface, which, in most
movie 5). The initial height degradation rate of 148.82
instances, is aligned in the shape of a cross (Figure
nm/min is calculated through fitting for the curve
2b). They are supposed to be the precursors of DNA
(see Figure S3 in the ESM). Furthermore, the DNA
box [37]. 3D DNA box origami is also yielded in
origami in 10 vol% serum is also observed. Because
Figure 2a. Analysis of the high resolution AFM
there is also a similar abrupt change in the beginning
images of individual particles revealed x and y
after >1 vol% serum addition, more information are
dimensions that are in good agreement with the
put into ESM (see Figure S4 in the ESM and
shape and dimensions of the designed DNA box as
Supplementary movie 6). As discussed above, the 3D
we have reported (see Figure 2d) [13, 15].
DNA box origami exhibit a visible degradation
After proving the successful synthesis of 3D
process in 0.1 vol% serum solution. The initial slope
DNA box, the stability of the 3D DNA box origami in
is about -21.01 calculated from the explinear fitting
serum was investigated using HS-AFM. At t=0s, the
curve for addition of 0.1 vol% serum in Figure 3c
imaging solution was exchanged with 0.1 vol%
(more information can be seen in Figure S5 in the
serum aqueous solution using a flow-through system
ESM). That means the initial height degradation
(the diluted liquid used is the 1×TAE/Mg2+ buffer).
speed of 3D DNA box origami after serum injection
One image was taken every 36s for the initial ten
is about 21.01 nm/min after 0.1 vol% serum additions.
images. And with the motivation to minimize the
The degradation behaviors of 3D DNA box origami
| www.editorialmanager.com/nare/default.asp
5
Nano Res.
in serum with different concentrations are further
raises the question of the difference behavior of 3D
confirmed by agarose gel electrophoresis experiment
DNA box origami between actual situation and test
(Figure 3d). After immersion in 0.1 vol% or 1vol%
conditions. Answering this could be important for
serum, the 3D DNA box origami do not run as a
understanding the mechanism by which 3D DNA
single band but is smeared throughout the lane: the
box origami can develop resistance to serum. The
appearance of products with smeared faster mobility
mica surface may have some stabilizing role for the
indicates that some of the 3D DNA box origami is
bottom side of box origami [29]. However, it is worth
digested by serum enzymes; the products with
noting that the damage of box origami in serum
smeared slower mobility indicate severe protein
solution may be due to its hollow structure. The
binding and maybe some degradation. In the case of
serum may firstly destroy its structural stability, and
0.01 vol% or 0.001 vol% serum addition, nearly the
height collapse is then happened, in which more than
entire sample of 3D DNA box origami remain in the
80% of the damages are completed (see Figure 3b).
gel well, as evidenced by their representative bands,
The stabilizing effect of mica surface almost has no
comparing the gels from the untreated sample.
influence on the structure of box origami. So it
Finally, the kinetics of degradation process of
indicates that there is same behavior of 3D DNA box
single 3D DNA box origami was investigated. To
origami
quantify the kinetics, we follow the change in the
situation and test conditions. Thus, the DNA box
height profiles along the middle line of the top
origami should have some essential modifications to
surface of single 3D DNA box origami in every frame
improve its stability. As discussed above, the most
of the image, as shown in Figure 4a. The change in
damages are happened in the collapse phase. So the
3D DNA box origami structure is visible ~36s after
modifications to increase the strength or number of
the addition of serum. Figure 4b shows the height
the hybridization linkers between two nearby sheets
variation of a single 3D DNA box origami as a
are supposed to be the option to improve the
function of time after injection of 0.1 vol% serum. The
structure stability of the box origami in the collapse
corresponding curve is fitted with a explinear
phase.
4 Conclusions
In conclusion, we have successfully investigated
the dynamics behavior of 3D DNA box origami in
serum with different concentration by monitoring the
changes in the nanostructure by a high-speed AFM
scanning system. The critical concentration of serum
for 3D DNA box origami degradation is about 0.1
vol%. The lifetime of 3D DNA box origami in 0.1
vol% serum is 62±26s. The digest process is a
combination of a rapid height collapse phase and a
slow degradation phase (which takes half an hour to
complete). And most of damages happen in the rapid
collapse phase. These results indicate that DNA box
origami should have some surface modifications to
increase its stability before its clinical applications.
Especially, to improve its structure stability in
collapse phase will produce a better effect. It is
noteworthy that this is the first report on the
real-time observation of a degradation process for 3D
function (see Figure S6 in ESM). Figure 4c shows the
snap-shots of the HS-AFM imaging of singe box
origami and schematics of the degradation events
after serum action. From the analysis for above
obtain data, we propose that the digest of the 3D
DNA box origami by 0.1 vol% serum under testing
condition is a two-stage process consisting of a
height collapse phase, in which the height of 3D
DNA box origami is suddenly decrease in less than
1.5min, followed by a slow degradation phase for 3D
DNA box origami, which can last from several
minutes to half an hour. The time to complete the
slow degradation phase is far longer than that to
complete the height collapse phase. This result
suggests that the bulk degradation rate is dominated
by the time it takes to complete the slow degradation
phase, rather than the height collapse phase. It also
after
serum addition
between
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
actual
Research
6
Nano Res.
DNA origami. We anticipate that our primary results
could pave the way for the direct observation of
various structural changes of origami, in real time, at
the nanometer level.
self-assembled from single-stranded DNA
tiles. Nature 2012, 485, 623-626.
[6]
Y.; Yan, H. DNA Origami with Complex
Curvatures in Three-Dimensional Space.
Acknowledgements
The authors acknowledge financial support from
iNANO through the Danish National Research
Foundation and the National Natural Science
Foundation of China to the Sino-Danish Center of
excellence on “The Self-assembly and Function of
Molecular Nanostructures on Surfaces”, the
Carlsberg Foundation, and the Villum Foundation.
The authors would like to thank National Natural
Science Foundation of China (No. 51003021), China
Postdoctoral Science Special Foundation (No.
201003420, No.20090460067).
Electronic
Supplementary
Material:
Supplementary material (Fitting for the height
Science 2011, 332, 342-346.
[7]
article at http://dx.doi.org/10.1007/s12274-***-****-*
(automatically inserted by the publisher).
2012, 338, 1177-1183.
[8]
Microscopy.
[9]
4, 2234-2240.
[10]
Maune,
H.
T.;Han,
Nanotechnology.
Accounts
in
of
Research 2014, 10.1021/ar500034y.
W.
origami templates. Nature Nanotechnology
2010, 5, 61-66.
[11]
Subramani, R.;Juul, S.;Rotaru, A.;Andersen,
F.
F.;Gothelf,
K.
V.;Mamdouh,
W.;Besenbacher, F.;Dong, M.; Knudsen, B. R.
A Novel Secondary DNA Binding Site in
Human Topoisomerase I Unravelled by
using a 2D DNA Origami Platform. ACS
Nano 2010, 4, 5969-5977.
[12]
Ke, Y. G.;Sharma, J.;Liu, M. H.;Jahn, K.;Liu,
Y.; Yan, H. Scaffolded DNA Origami of a
DNA Tetrahedron Molecular Container.
DNA
Chemical
M.;Goddard,
R.
two-dimensional geometries using DNA
Tørring, T.;Helmig, S.;Ogilby, P. R.; Gothelf,
Oxygen
P.;Barish,
Self-assembly of carbon nanotubes into
Torring, T.;Voigt, N. V.;Nangreave, J.;Yan,
Singlet
S.
A.;Rothemund, P. W. K.; Winfree, E.
H.; Gothelf, K. V. DNA origami: a quantum
[5]
Deng, Z.;Pal, S.;Samanta, A.;Yan, H.; Liu, Y.
nanoheterostructures. Chemical Science 2013,
440, 297-302.
V.
48,
semiconductor nanowires for multiplex
nanoscale shapes and patterns. Nature 2006,
K.
2009,
DNA functionalization of colloidal II-VI
Rothemund, P. W. K. Folding DNA to create
Chemical Society Reviews 2011, 40, 5636-5646.
Edition
8870-8873.
DNA origami design of dolphin-shaped
leap for self-assembly of complex structures.
T.
Angewandte
Chemie-International
Andersen, E. S.;Dong, M. D.;Nielsen, M.
2, 1213-1218.
R.;Sobey,
as a Nanoscopic Ruler for Super-Resolution
W.;Gothelf, K. V.;Besenbacher, F.; Kjems, J.
[4]
C.;Jungmann,
L.;Simmel, F. C.; Tinnefeld, P. DNA Origami
M.;Jahn, K.;Lind-Thomsen, A.;Mamdouh,
[3]
Steinhauer,
D.;Bockrath,
structures with flexible tails. Acs Nano 2008,
Structures
Self-Assembled from DNA Bricks. Science
References
[2]
Ke, Y.;Ong, L. L.;Shih, W. M.; Yin, P.
Three-Dimensional
variation curve of 3D DNA box origami after different
concentration serum injection and corresponding
movies) is available in the online version of this
[1]
Han, D.;Pal, S.;Nangreave, J.;Deng, Z.;Liu,
Nano Letters 2009, 9, 2445-2447.
[13]
Wei, B.;Dai, M.; Yin, P. Complex shapes
| www.editorialmanager.com/nare/default.asp
Zadegan, R. M.;Jepsen, M. D. E.;Thomsen,
K.
E.;Okholm,
A.
H.;Schaffert,
D.
H.;Andersen, E. S.;Birkedal, V.; Kjems, J.
7
Nano Res.
Construction of a 4 Zeptoliters Switchable
Journal of the American Chemical Society 2010,
3D DNA Box Origami. Acs Nano 2012, 6,
132, 13545-13552.
10050-10053.
[14]
[22]
Hung, A. M.;Micheel, C. M.;Bozano, L.
L.;Arnbjerg, J.;Ogilby, P. R.;Kjems, J.;Mokhir,
D.;Osterbur, L. W.;Wallraff, G. M.; Cha, J. N.
A.;Besenbacher, F.; Gothelf, K. V. Single
Large-area spatially ordered arrays of gold
Molecule Atomic Force Microscopy Studies
nanoparticles directed by lithographically
of Photosensitized Singlet Oxygen Behavior
confined
on a DNA Origami Template. Acs Nano 2010,
DNA
origami.
Nature
Nanotechnology 2010, 5, 121-126.
[15]
4, 7475-7480.
Andersen, E. S.;Dong, M.;Nielsen, M.
M.;Jahn,
K.;Subramani,
W.;Golas,
M.
[17]
M.;Sander,
[20]
Origami
V.;Besenbacher, F.;Gothelf, K. V.; Kjems, J.
Fabrication. Acs Nano 2011, 5, 2240-2247.
[24]
for
Nanoelectronic
Circuit
Ding, B. Q.;Deng, Z. T.;Yan, H.;Cabrini,
a controllable lid. Nature 2009, 459, 73-U75.
S.;Zuckermann, R. N.; Bokor, J. Gold
Keum, J.-W.; Bermudez, H. Enhanced
Nanoparticle Self-Similar Chain Structure
resistance
Organized by DNA Origami. Journal of the
of
DNAnanostructures
enzymatic
digestion.
Communications
2009,
to
Chemical
10.1039/b917661f,
American Chemical Society 2010, 132, 3248-+.
[25]
Mo, Y.;Turner, K. T.; Szlufarska, I. Friction
7036-7038.
laws at the nanoscale. Nature 2009, 457,
Walsh, A. S.;Yin, H.;Erben, C. M.;Wood, M.
1116-1119.
[26]
Mei, Q.
A.;Wei, X.
X.;Su, F.
Y.;Liu,
to Mammalian Cells. ACS Nano 2011, 5,
Y.;Youngbull, C.;Johnson, R.;Lindsay, S.;Yan,
5427-5432.
H.; Meldrum, D. Stability of DNA Origami
Li, J.;Pei, H.;Zhu, B.;Liang, L.;Wei, M.;He,
Nanoarrays in Cell Lysate. Nano Letters 2011,
Y.;Chen, N.;Li, D.;Huang, Q.; Fan, C.
11, 1477-1482.
Self-Assembled
Multivalent
Nanostructures
for
DNA
[27]
Noninvasive
Delivery
Conway, J. W.;McLaughlin, C. K.;Castor, K.
J.; Sleiman, H. DNA nanostructure serum
of
stability: greater than the sum of its parts.
Immunostimulatory CpG Oligonucleotides.
Chemical
ACS Nano 2011, 5, 8783-8789.
1172-1174.
Fu, J.; Yan, H. Controlled drug release by a
[28]
Communications
2013,
49,
Song, J.;Zhang, Z.;Zhang, S.;Liu, L.;Li,
nanorobot. Nat Biotech 2012, 30, 407-408.
Q.;Xie, E.;Gothelf, K. V.;Besenbacher, F.;
Schreiber, R.;Kempter, S.;Holler, S.;Schuller,
Dong, M. Isothermal Hybridization Kinetics
V.;Schiffels, D.;Simmel, S. S.;Nickels, P. C.;
of DNA Assembly of Two-Dimensional
Liedl, T. DNA Origami-Templated Growth
DNA Origami. Small 2013, 9, 2954-2959.
of Arbitrarily Shaped Metal Nanoparticles.
[21]
Harb, J. N. Metallization of Branched DNA
H.;Oliveira, C. L. P.;Pedersen, J. S.;Birkedal,
Intracellular
[19]
Liu, J. F.;Geng, Y. L.;Pound, E.;Gyawali,
S.;Ashton, J. R.;Hickey, J.;Woolley, A. T.;
B.;Stark,
J. A.; Turberfield, A. J. DNA Cage Delivery
[18]
[23]
R.;Mamdouh,
Self-assembly of a nanoscale DNA box with
[16]
Helmig, S.;Rotaru, A.;Arian, D.;Kovbasyuk,
[29]
Song, J.;Arbona, J.-M.;Zhang, Z.;Liu, L.;Xie,
Small 2011, 7, 1795-1799.
E.;Elezgaray,
J.;Aime,
Li, Z.;Liu, M. H.;Wang, L.;Nangreave, J.;Yan,
V.;Besenbacher,
H.; Liu, Y. Molecular Behavior of DNA
Visualization
Origami in Higher-Order Self-Assembly.
Response of a DNA Origami. Journal of the
F.;
of
J.-P.;Gothelf,
Dong,
M.
Transient
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
K.
Direct
Thermal
Research
8
Nano Res.
American
Chemical
Society
2012,
134,
[37]
9844-9847.
[30]
[31]
Ando,
Andersen, E. S.;Dong, M.;Nielsen, M.
M.;Jahn,
T.;Uchihashi,
T.;
Fukuma,
T.
W.;Golas,
K.;Subramani,
M.
R.;Mamdouh,
M.;Sander,
B.;Stark,
High-speed atomic force microscopy for
H.;Oliveira, C. L. P.;Pedersen, J. S.;Birkedal,
nano-visualization
dynamic
V.;Besenbacher, F.;Gothelf, K. V.; Kjems, J.
biomolecular processes. Progress in Surface
of
Self-assembly of a nanoscale DNA box with
Science 2008, 83, 337-437.
a controllable lid. Nature 2009, 459, 73-76.
Ando, T.;Kodera, N.;Takai, E.;Maruyama,
D.;Saito, K.; Toda, A. A high-speed atomic
force microscope for studying biological
FIGURES.
macromolecules. Proceedings of the National
Academy of Sciences 2001, 98, 12468-12472.
[32]
Rajendran,
A.;Endo,
Sugiyama,
H.
Direct
M.;Hidaka,
and
K.;
Real-Time
Observation of Rotary Movement of a DNA
Nanomechanical Device. Journal
American
Chemical
of the
Society
2012,
10.1021/ja310454k.
[33]
Endo, M.;Katsuda, Y.;Hidaka, K.; Sugiyama,
H. Regulation of DNA Methylation Using
Different Tensions
of
Double Strands
Constructed
a
Defined
in
Nanostructure. Journal
DNA
of the American
Chemical Society 2010, 132, 1592-1597.
[34]
Sannohe, Y.;Endo, M.;Katsuda, Y.;Hidaka,
K.; Sugiyama, H. Visualization of Dynamic
Conformational
Switching
of
the
G-Quadruplex in a DNA Nanostructure.
Journal of the American Chemical Society 2010,
132, 16311-16313.
[35]
Rajendran,
Sugiyama,
A.;Endo,
H.
Direct
M.;Hidaka,
and
K.;
Real-Time
Observation of Rotary Movement of a DNA
Nanomechanical Device. Journal
American
Chemical
Society
2013,
of the
135,
1117-1123.
[36]
Fantner, G. E.;Barbero, R. J.;Gray, D. S.;
Belcher, A. M. Kinetics of antimicrobial
peptide activity measured on individual
bacterial cells using high-speed atomic
force microscopy. Nature Nanotechnology
2010, 5, 280-285.
Figure 1 Small AFM cantilevers for high-speed AFM. a,
SEM image of a small cantilever. The inset optical
images compare a normal lever (left) and the smaller
cantilever (right) used for AFM imaging in fluid at the
same magnification. b, Thermal noise power spectra of
regular and smaller cantilevers. In air (red solid line),
the first resonance frequency of the small cantilever is
~240 kHz. In aqueous solution this drops to 60~90 kHz
(red dashed line). The inset shows the thermal noise
power spectra of a normal one (PNP-TR-TL-Au,
Olympus) with resonance frequencies of ~20 kHz in air
(blue solid line) and ~4 kHz in aqueous solution (blue
| www.editorialmanager.com/nare/default.asp
9
Nano Res.
dashed line).
Figure 2 a, HS-AFM image of the 3D DNA box
origami. b, HS-AFM image of the 2D DNA sheet
origami. c, HS-AFM image of the single 3D DNA box
origami. Samples were prepared in 20mM Tris-HCl
buffer (pH 7.6) containing 10 mM Mg2+, and the images
were recorded in the same buffer. Scan speed: 8 line/s;
image size: 2×2 μm. d, Height distribution of 2D DNA
sheet origami (light gray histogram) and the 3D DNA
box origami (dark gray histogram).
Figure 3 a, Successive HS-AFM images of 3D DNA
box origami at the critical point in time. b, Survaial
persent variation of the 3D DNA box origami as a
function of time after injection of serum. c, Bulk
measurement of serum activity with different injection
dose. d, Agarose gel electrophoresis of 3D DNA box
origami in serum: lane 1, 1000 bp DNA ladder; lane 2,
3D DNA box origami only; lane 3, 3D DNA box
origami in 1 vol% serum; lane 4, 3D DNA box origami
in 0.1 vol% serum; lane 5, 3D DNA box origami in 0.01
vol% serum; lane 6, 3D DNA box origami in 0.001
vol% serum.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
10
Nano Res.
Figure 4 a, Cross-sections of 3D DNA box origami showing the time progression of the structure variation. Each
slice represents data extracted from one image in the full time series. b, Height variation of a single 3D DNA box
origami as a function of time after injection of 0.1 vol% serum. The gray dash line divided the fitting curve into two
parts. The left part is belonging to the collapse phase, and the right part is the slow degradation phase. In addition,
the x error is the time for every image. It is found that the height variation of origami box is not sensitive to the time
after 4.5min. So it is reasonably to double the acquisition time for images every ten images. c, Snapshots of the
HS-AFM imaging and schematics of the degradation events after serum action.
| www.editorialmanager.com/nare/default.asp
11
Nano Res.
COVER FIGURE:
DNA origami-box degradation kinetics
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
Nano Res.
Electronic Supplementary Material
Serum Induced Degradation of 3D DNA Box Origami
Observed by High Speed Atomic Force Microscope
Zaixing Jiang1,2,†, Shuai Zhang2,†, Chuanxu Yang2, Jørgen Kjems 2, Yudong Huang1,*, Flemming
Besenbacher 2, Mingdong dong2,*
Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)
25
20
Hz (nm)
15
10
5
0
0
10
20
30
40
50
Time after serum injection (min)
Figure S1 Linear fitting for the height variation curve of 3D DNA box origami after 0.001 vol% serum injection.
The fitting formula is y=a+bx , where a=19.72622, b=-0.00631.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
Nano Res.
25
20
Hz (nm)
15
10
5
0
0
10
20
30
40
50
Time after serum injection (min)
Figure S2 Linear fitting for the height variation curve of 3D DNA box origami after 0.01 vol% serum injection.
The fitting formula is y=a+bx, where a=20.20739, b=-0.01548.
25
20
Hz (nm)
15
10
5
0
0
10
20
30
40
50
Time after serum injection (min)
Figure S3 Explinear fitting for the height variation curve of 3D DNA box origami after 1 vol% serum injection.
The fitting formula is y=a·exp(-x/b)+c+dx, where a=17.81782, b=0.11972, c=1.08128 d=-0.00136. The initial
degradation speed is a/b, i.e. the first derivative for the exponential part of the fitting formula.
| www.editorialmanager.com/nare/default.asp
Nano Res.
20
Hz (nm)
15
10
5
0
0
10
20
30
40
50
Time after serum injection (min)
Figure S4 Logistic fitting for the height variation curve of 3D DNA box origami after 10 vol% serum injection.
The fitting formula is y=a·exp(-x/b)+c+dx, where a=18.4837, b=0.10759, c=0 d=0. The initial degradation speed is
a/b, i.e. the first derivative for the exponential part of the fitting formula.
25
20
Hz (nm)
15
10
5
0
0
10
20
30
40
50
Time after serum injection (min)
Figure S5 Explinear fitting for the height variation curve of 3D DNA box origami after 0.1 vol% serum injection.
The fitting formula is y=a·exp(-x/b)+c+dx, where a=9.8228; b=0.46743; c=8.06492; d=-0.16239. The initial
degradation speed is a/b, i.e. the first derivative for the exponential part of the fitting formula.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
Nano Res.
18
16
Hm (nm)
14
12
10
8
6
0
5
10
15
20
25
30
35
Time after addition of serum (min)
Figure S6 Explinear fitting for the height variation curve of single 3D DNA box origami after 0.1 vol% serum
injection. The fitting formula is y=a·exp(-x/b)+c+dx, where a=11.21636; b=0.30384; c=6.74917; d=-0.01131. The
initial degradation speed is a/b, i.e. the first derivative for the exponential part of the fitting formula.
Supplementary movie 1, the movie of the 3D DNA box obtained by HS-AFM.
| www.editorialmanager.com/nare/default.asp
Nano Res.
Supplementary movie 2, the degradation movie of the 3D DNA box in 0.1 vol% serum obtained by HS-AFM.
Supplementary movie 3, the degradation movie of the 3D DNA box in 0.01 vol% serum obtained by HS-AFM.
Supplementary movie 4, the degradation movie of the 3D DNA box in 0.001 vol% obtained by HS-AFM.
Supplementary movie 5, the degradation movie of the 3D DNA box in 1 vol% serum obtained by HS-AFM.
www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano
Research
Nano Res.
Supplementary movie 6, the degradation movie of the 3D DNA box in 10 vol% serum obtained by HS-AFM.
| www.editorialmanager.com/nare/default.asp