Serum Induced Degradation of 3D DNA Box Origami Serum Induced Degradation of 3D DNA Box Origami Observed by High Speed Atomic Force Microscope Zaixing Jiang1,2,†, Shuai Zhang2,†,

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

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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





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+

http://www.lifetechnologies.com/order/catalog/product/10100139

http://www.lifetechnologies.com/order/catalog/product/10100139

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

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,


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


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


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

http://www.nanoworld.com/pyrex-nitride-gold-coated-triangular-silicon-nitride-tipless-cantilever-afm-tip-pnp-tr-tl-au


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





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

)


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

)


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

)


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

)


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.

Documents

Serum Induced Degradation of 3D DNA Box Origami Serum Induced Degradation of 3D DNA Box Origami Observed by High Speed Atomic Force Microscope Zaixing Jiang1,2,†, Shuai Zhang2,†,