Serum Induced Degradation of 3D DNA Box Origami Serum Induced Degradation of 3D DNA Box Origami...
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Nano Res
1
Serum Induced Degradation of 3D DNA Box Origami
Observed by High Speed Atomic Force Microscope
Zaixing Jiang1,2,†, Shuai Zhang2,†, Chuanxu Yang2, Jørgen Kjems2, Yudong Huang1,*, Flemming
Besenbacher2, Mingdong dong2,*
Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0724-z
http://www.thenanoresearch.com on January 28, 2015
© Tsinghua University Press 2015
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Nano Research
DOI 10.1007/s12274-015-0724-z
TABLE OF CONTENTS (TOC)
Serum Induced
Degradation of 3D
DNA Box Origami
Observed by High
Speed Atomic Force
Microscope
Zaixing Jianga,b, Shuai
Zhang b, Chuanxu
Yang b, Jørgen Kjems
b, Yudong Huanga,*,
Flemming
Besenbacher b,
Mingdong dong b ,*
a Department of
Polymer Science and
Technology, School of
Chemical Engineering
and Technology,
Harbin Institute of
Technology, Harbin
150001, People’s
Republic of China
b Interdisciplinary
Nanoscience Center
(iNANO), Aarhus
University, DK-8000,
Aarhus C, Denmark
Insert your TOC graphics here.
The degradation kinetics of 3D DNA box origami in serum using high speed atomic force
microscope has been demonstrated. Our findings are valuable for the further modifications to
improve the biocompatibility of DNA nanostructures in future applications.
Provide the authors’ webside if possible.
Author 1, webside 1
Author 2, webside 2
Serum Induced Degradation of 3D DNA Box Origami
Observed by High Speed Atomic Force Microscope
Zaixing Jiang1,2,†, Shuai Zhang2,†, Chuanxu Yang2, Jørgen Kjems2, Yudong Huang1,*, Flemming
Besenbacher2, Mingdong dong2,*
Received: day month year
Revised: day month year
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
ABSTRACT
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.
1 Introduction
As being one recently developed high efficient
self-assembly technique, the DNA origami is one
radically increased subgroup of DNA
nanotechnology [1-4]. By folding a long single DNA
strand into arbitrary shapes with hundreds of
synthetic staple strands, DNA origami has been
proved to form 2D and 3D nanostructure, from
simplicity to complicity, with high yields and
accuracy [5-7]. These nanostructures have many
applications, such as serving as nanoscale rulers for
single molecule imaging [8], templates for the
nanowire growth [9, 10], aid in the molecular
structure determination [11, 12], and new platforms
for genomics applications [13, 14]. 3D DNA box [15],
which was first designed and synthesized in 2009, is
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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 nm3
(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+
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3 Nano Res.
buffer. The FBS was carefully injected into the AFM
cell by our homemade injection system. The injection
system was composed by syringe (1.0ml), syringe
pump (Aladdin-1000, Word Precision Instruments
Programmable), rubber tube (Φ1.0mm), needle (4.5#)
and needle holder. In order to avoid the bubble
interference, the FBS was full fill the rubber tube and
needle. The AFM was fistly begin to work, and after
it had got at least two images, the injection system
begin to work. The FBS was injected automatically by
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.
The height of the box is calculated from software of
SPIPTM.
2.4 High-speed Atomic force microscopy
AFM images were obtained using Dimension
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
frequency in liquid with only a 0.4-1.2 N/m force
constant. The Silicon tip has an extremely sharp
with 5 nm tip radius, making it ideal for imaging a
wide variety of hard and soft materials. All
FastScan cantilevers have less that 3 degrees of
cantilever bend.
During the experiment, the sample (1 μL) was
adsorbed onto a freshly cleaved mica plate (ϕ12
mm, Bruker, CA) for 1min at room temperature,
and then 1×TAE/Mg2+ buffer (40 mM Tris pH:8.0, 2
mM EDTA pH:8.0, 12.5 mM MgAc2) was added. All
measurements were performed in tapping mode in
fluid. The experiment temperature is about 25℃.
Flow-through fluid exchange was achieved using a
dual syringe pump. Height, amplitude and phase
signals were recorded for both trace and retrace.
The data were processed using NanoScope
AnalysisTM, SPIPTM and Cinema 4DTM, using
standard modification commands applied over the
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]:
and
Where E and ρ are Young’s modulus and the
density of the used material, respectively. To attain a
high resonant frequency and a small spring constant
simultaneously, cantilevers with small dimensions
must be fabricated. In addition to the advantage in
achieving a high imaging rate, small cantilevers have
other advantages, such as lower noise density, less
affected by thermal noise and high sensitivity to the
gradient of the force exerted between the tip and the
sample. Thus, only small cantilever can balance the
high speed and low force on sample. In this work,
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4 Nano Res.
the small cantilevers, made of silicon nitride and
coated with gold of about 20 nm thickness, are used.
They have resonance frequencies in liquid at ~80 kHz
and spring constants between 0.4 and 1.2 nN/nm.
The higher resonance frequency, the lower mass, and
smaller spring constant enable the imaging speed in
tapping mode to be increased while keeping the
imaging forces on the 3D DNA box origami small.
Figure 1a shows a scanning electron microscopy
(SEM) image of the small cantilever used in this
research. The inset is a comparison between a
conventional cantilever and the small one. Figure 1b
shows the thermal spectra of the two types of
cantilevers. The resonance frequency of the small
cantilever is almost 20 times higher than that of the
conventional cantilever in fluid.
Before serum was injected, the morphology of
3D DNA box was characterized in advance. The
sample was prepared and adsorbed onto a freshly
cleaved mica substrate. After adding the observation
buffer containing Mg2+, the high-speed AFM
experiment was started with a larger scan area to
investigate morphology of 3D DNA box origami.
These results are shown in Figure 2 (for the full series
see Supplementary movie 1). As seen, there remains
lots of DNA sheets on mica surface, which, in most
instances, is aligned in the shape of a cross (Figure
2b). They are supposed to be the precursors of DNA
box [37]. 3D DNA box origami is also yielded in
Figure 2a. Analysis of the high resolution AFM
images of individual particles revealed x and y
dimensions that are in good agreement with the
shape and dimensions of the designed DNA box as
we have reported (see Figure 2d) [13, 15].
After proving the successful synthesis of 3D
DNA box, the stability of the 3D DNA box origami in
serum was investigated using HS-AFM. At t=0s, the
imaging solution was exchanged with 0.1 vol%
serum aqueous solution using a flow-through system
(the diluted liquid used is the 1×TAE/Mg2+ buffer).
One image was taken every 36s for the initial ten
images. And with the motivation to minimize the
disruption of 3D DNA box origami structure caused
by AFM tip, the acquisition time will be doubled
after every ten images. Four images obtained at the
critical point in time are shown in Figure 3a (for the
full series see Supplementary movie 2). It is evident
that most of 3D DNA boxes have a similar respond
time to the addition of serum solution. Figure 3b
shows the survival percent values for the 3D DNA
box origami as a function of time after the addition of
serum. Time of degradation is highly variable, with
an average of 62±26s. Serum
concentration-dependent experiments are also
executed with lower (0.01 vol% and 0.001 vol%) and
higher (1.0 vol%) serum concentrations. The
corresponding results are shown in Figure 3c. At
lower serum concentration, no degradation of the 3D
DNA box origami is observed (for the full series see
supplementary movie 3 and 4). The slope of the two
plots almost has no change, i.e. the degradation
speed approaching zero (see Figure S1 and S2 in the
Electronic Supplementary Material (ESM)).
Conversely, at 1 vol% serum addition, an abrupt
change is happened in the beginning, i.e. all of 3D
DNA boxes are destroyed immediately after the
serum addition (for the full series see Supplementary
movie 5). The initial height degradation rate of 148.82
nm/min is calculated through fitting for the curve
(see Figure S3 in the ESM). Furthermore, the DNA
origami in 10 vol% serum is also observed. Because
there is also a similar abrupt change in the beginning
after >1 vol% serum addition, more information are
put into ESM (see Figure S4 in the ESM and
Supplementary movie 6). As discussed above, the 3D
DNA box origami exhibit a visible degradation
process in 0.1 vol% serum solution. The initial slope
is about -21.01 calculated from the explinear fitting
curve for addition of 0.1 vol% serum in Figure 3c
(more information can be seen in Figure S5 in the
ESM). That means the initial height degradation
speed of 3D DNA box origami after serum injection
is about 21.01 nm/min after 0.1 vol% serum additions.
The degradation behaviors of 3D DNA box origami
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5 Nano Res.
in serum with different concentrations are further
confirmed by agarose gel electrophoresis experiment
(Figure 3d). After immersion in 0.1 vol% or 1vol%
serum, the 3D DNA box origami do not run as a
single band but is smeared throughout the lane: the
appearance of products with smeared faster mobility
indicates that some of the 3D DNA box origami is
digested by serum enzymes; the products with
smeared slower mobility indicate severe protein
binding and maybe some degradation. In the case of
0.01 vol% or 0.001 vol% serum addition, nearly the
entire sample of 3D DNA box origami remain in the
gel well, as evidenced by their representative bands,
comparing the gels from the untreated sample.
Finally, the kinetics of degradation process of
single 3D DNA box origami was investigated. To
quantify the kinetics, we follow the change in the
height profiles along the middle line of the top
surface of single 3D DNA box origami in every frame
of the image, as shown in Figure 4a. The change in
3D DNA box origami structure is visible ~36s after
the addition of serum. Figure 4b shows the height
variation of a single 3D DNA box origami as a
function of time after injection of 0.1 vol% serum. The
corresponding curve is fitted with a explinear
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
raises the question of the difference behavior of 3D
DNA box origami between actual situation and test
conditions. Answering this could be important for
understanding the mechanism by which 3D DNA
box origami can develop resistance to serum. The
mica surface may have some stabilizing role for the
bottom side of box origami [29]. However, it is worth
noting that the damage of box origami in serum
solution may be due to its hollow structure. The
serum may firstly destroy its structural stability, and
height collapse is then happened, in which more than
80% of the damages are completed (see Figure 3b).
The stabilizing effect of mica surface almost has no
influence on the structure of box origami. So it
indicates that there is same behavior of 3D DNA box
origami after serum addition between actual
situation and test conditions. Thus, the DNA box
origami should have some essential modifications to
improve its stability. As discussed above, the most
damages are happened in the collapse phase. So the
modifications to increase the strength or number of
the hybridization linkers between two nearby sheets
are supposed to be the option to improve the
structure stability of the box origami in the collapse
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
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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.
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
variation curve of 3D DNA box origami after different
concentration serum injection and corresponding
movies) is available in the online version of this
article at http://dx.doi.org/10.1007/s12274-***-****-*
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FIGURES.
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
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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.
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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.
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COVER FIGURE:
DNA origami-box degradation kinetics
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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 Kjems2, Yudong Huang1,*, Flemming
Besenbacher2, Mingdong dong2,*
Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)
0 10 20 30 40 50
0
5
10
15
20
25
Hz (
nm
)
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.
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0 10 20 30 40 50
0
5
10
15
20
25
Hz (
nm
)
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.
0 10 20 30 40 50
0
5
10
15
20
25
Hz (
nm
)
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.
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0 10 20 30 40 50
0
5
10
15
20
Hz (
nm
)
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.
0 10 20 30 40 50
0
5
10
15
20
25
Hz (
nm
)
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.
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0 5 10 15 20 25 30 35
6
8
10
12
14
16
18
Hm (
nm
)
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.
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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.
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Supplementary movie 6, the degradation movie of the 3D DNA box in 10 vol% serum obtained by HS-AFM.