Frame-guided assembly of vesicles with programmed geometry and dimensions

Article abstract of DOI:10.1002/anie.201310715

In molecular self-assembly molecules form organized structures or patterns. The control of the self-assembly process is an important and challenging topic. Inspired by the cytoskeletal-membrane protein lipid bilayer system that determines the shape of euk

Full text of DOI:10.1002/anie.201310715

Angewandte
Chemie
DOI: 10.1002/anie.201310715
Vesicle Self-Assembly
Hot Paper
Frame-Guided Assembly of Vesicles with Programmed Geometry and
Dimensions**
Yuanchen Dong, Yawei Sun, Liying Wang, Dianming Wang, Tao Zhou, Zhongqiang Yang,
Zhong Chen, Qiangbin Wang, Qinghua Fan, and Dongsheng Liu*
Abstract: In molecular self-assembly molecules form organ-
ized structures or patterns. The control of the self-assembly
process is an important and challenging topic. Inspired by the
cytoskeletal-membrane protein lipid bilayer system that deter-
mines the shape of eukaryotic cells, we developed a frame-
guided assembly process as a general strategy to prepare
heterovesicles with programmed geometry and dimensions.
This method offers greater control over self-assembly which
may benefit the understanding of the formation mechanism as
well as the functions of the cell membrane.
The principle of frame-guided assembly is illustrated in
Scheme 1. Discontinuous, pre-positioned leading hydropho-
bic groups (LHGs) outline the fringe of the designed
structures and guide other amphiphilic molecules to fill in
the gaps between LHGs, finally leading to the formation of
heterovesicles with designed shape and size. Gold nano-
particles (AuNPs) functionalized with thiolated, 20nucleotide
(20nt) single-stranded DNA (ssDNA)[5] are used as the
foundation of the frame. The LHGs, covalently bound to the
complementary 20nt ssDNA, are directed to the correspond-
ing positions by DNA hybridization. As part of our strategy
a given amount of thiolated 6nt ssDNA was mixed with the
20nt ssDNA before incubation with the AuNPs; due to
Amphiphilic molecules spontaneously assemble into spher-
ical micelles, vesicles, or other symmetric forms in water, with
the shape of the assemblies determined by the laws of
thermodynamics and their intrinsic molecular properties.[1]
Over the past several decades there has been a concerted
effort to enhance our understanding of and control over the
assembly process.[2] However, obtaining amphiphilic assem-
blies with customized shapes and sizes still presents a signifi-
cant challenge in this field.[3] Inspired by the cytoskeletal-
membrane protein lipid bilayer system that determines the
shape of eukaryotic cells,[4] we developed a frame-guided
assembly process as a general strategy to prepare customized
heterovesicles. Our method offers greater control over self-
assembly which may benefit the understanding of the
formation mechanism as well as the functions of the cell
membrane.
[*] Y. Dong, Dr. Y. Sun, D. Wang, T. Zhou, Prof. Dr. Z. Yang,
Prof. Dr. D. Liu
Key Laboratory of Organic Optoelectronics & Molecular Engineer-
ing of the Ministry of Education
Department of Chemistry, Tsinghua University
Beijing 100084 (China)
E-mail: liudongsheng@tsinghua.edu.cn
Dr. L. Wang, Prof. Dr. Q. Fan
Beijing National Laboratory for Molecular Sciences
Institute of Chemistry and Graduate School
Chinese Academy of Sciences (China)
Z. Chen, Prof. Dr. Q. Wang
Division of Nanobiomedicine and i-Lab
Suzhou Institute of Nano-Tech and Nano-Bionics
Chinese Academy of Sciences (China)
[**] We thank the National Basic Research Program of China (973
program, No. 2013CB932803), the National Natural Science
Foundation of China (Nos. 91027046, 21121004), and the NSFC-
DFG joint project TRR61 for financial support.
Scheme 1. Illustration of the frame-guided assembly process. The
AuNPs are modified with 20nt and 6nt ssDNA. DDOEG is anchored
to AuNPs by hybridization with DNA. Upon the addition of G2Cl-18,
the LHGs guide G2Cl-18 to fill in the gaps between LHGs by
interactions with the hydrophobic dendron domain.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310715.
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electrostatic repulsion the longer ssDNA should be dispersed
by the shorter ssDNA. Thus, the LHGs are evenly and
discontinuously distributed along the fringe of the frame.[6]
Subsequently, other types of amphiphilic molecules are
introduced to fill in the gaps between LHGs and ultimately
heterovesicles are formed whose shape and size are deter-
mined by the frame design.
precipitation (Figure 1c), suggesting that the aggregation is
due to the positioning of LHGs by DNA hybridization. Based
on these observations we present a possible structure of the
sediment seen in Figure 1a: the interactions between LHGs
are amplified by multiple interacting sites[9] which in turn
cause aggregation of the frames resulting in precipitation.
Here, the frames associate through hydrophobic interactions
between LHGs such that the AuNPs between the frames are
drawn close enough together to account for the red shift
observed in the UV/Vis spectrum. Then, the additional G2Cl-
18 molecules diffuse into the hydrophobic region of the LHGs
eliminating the occurrence of multiple interactions among the
frames and ultimately resulting in the formation of hetero-
vesicles along the frame (Figure 1b).
To confirm this mechanism we examined the morphology
of the assemblies by transmission electron microscopy
(TEM). In Figure 2a,b there are obvious gaps between the
AuNPs in the precipitates that can be attributed to the
presence of DNA double helices and LHG aggregates, as
illustrated in Figure 1a. In comparison, aggregates of unmodi-
fied AuNPs induced by high salt concentration do not display
gaps between particles. This result indicates that the expected
frame structures assemble correctly, leading to the observed
aggregation. After the addition of G2Cl-18, only well-dis-
persed AuNPs were observed; a layer of organic molecules
can be seen around the AuNPs where the samples were
negatively stained by uranyl acetate (Figure 2c). These results
confirm that an organic vesicle indeed formed as designed. To
We considered several factors when selecting LHGs,
including molecular structure, size, and hydrophobicity; most
importantly, the molecules resulting after connection to DNA
must not self-aggregate at the concentration required for
hybridization with DNA-modified AuNPs. After systematic
optimization we chose the poly(aryl ether) dendron as
a model LHG, where one end is covalently bound to the
DNA used to anchor the LHG to the AuNPs and the other
dendritic end is connected to eight oligo(ethylene glycol)
(OEG) tails to suppress self-aggregation.[7] The DNA-Den-
OEG complex is referred to as DDOEG, and in our
experiments no assemblies of DDOEG in solutions with
a concentration of 10 mm is evident by transmission electron
microscopy (TEM) and dynamic light scattering (DLS). A
second-generation poly(benzyl ether) dendron peripherally
functionalized with dichlorophenyl groups and connected to
an 18nt ssDNA (G2Cl-18) was selected to serve as the
amphiphilic molecule to fill in the gaps between LHGs.[8]
In a typical experiment, 13nm AuNPs (30 nm) were
modified with 20nt and 6nt ssDNA (details in Table S1 in the
Supporting Information) at a ratio of 1:1, and were sub-
sequently incubated with 5 mm DDOEG at 48C for 3 h. As
shown in Figure 1a, precipitation was observed and the UV/
Vis absorption spectra exhibited a red shift (see Figure S5 in
the Supporting Information). Within 10 min of the addition of
5 mm G2Cl-18 the precipitate disappeared and the solution
became clear again as shown in Figure 1b. Further, the
absorption wavelength returned to 518 nm, suggesting that
the AuNPs had switched back to the initial monodispersed
state.[5] As a control, unmodified AuNPs were incubated with
DDOEG under the same conditions with no observed
Figure 1. Characterization of the frame-guided assembly process. a) A
mixture of 30 nm DNA-functionalized 13nm AuNPs and 5 mm DDOEG
was incubated in buffer (0.5ꢀTBE, 50 mm NaCl) at 48C for 3 h and
a precipitate formed. b) After removal of the supernatant an amphi-
philic molecule, G2Cl-18, was added to the solution at a final concen-
tration of 5 mm and the precipitate subsequently disappeared.
c) Unmodified 13nm AuNPs were incubated with DDOEG under the
same conditions as in (a). The solution remained clear and no
precipitate was evident.
Figure 2. TEM analysis of the assemblies. a) Precipitates (as shown in
Figure 1a), without negative stain. b) Magnification of the area out-
lined in red in (a). c) Heterovesicles organized by 13nm spherical
frames. d) Heterovesicles organized by rod-shapes frames. e) AFM
image of DNA-functionalized 13nm AuNPs. f) AFM image of the
heterovesicles. In (c,d) the samples are negatively stained and the
organic molecules appear as coronas around the AuNPs/AuNRs. The
scale bars correspond to 100 nm.
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further confirm that the organic vesicles are formed from
G2Cl-18 molecules inserting into the gaps between LHGs,
5nm AuNPs modified with DNA sequences complementary
to the DNA domain of the G2Cl-18 molecules were incubated
with the assembled vesicles. These 5nm AuNPs could
specifically attach to the assemblies while unmodified
AuNPs do not (see Figure S3 in the Supporting Information).
This verifies that G2Cl-18 molecules are successfully guided
by the frame to form the desired heterovesicles. We also
employed atomic force microscopy (AFM) to investigate the
morphology of the assemblies. As shown in Figure 2e,f, the
height of assemblies is considerably greater than that of
DNA-modified AuNPs, which further confirms the formation
of assemblies.
In order to demonstrate the modularity of the frame-
guided assembly process, we changed the components of the
frame and studied their assembly by dynamic light scattering
(DLS). Firstly, we replaced the 13nm AuNPs with 30nm
AuNPs and observed similar phenomena, except the size of
the assemblies increase accordingly (see Figure S2 in the
Supporting Information). Secondly, we changed only the
length of double-stranded DNA (dsDNA) in the frame and
the results are shown in Figure 3: 1) After the attachment of
the final morphology of the heterovesicles, which is analogous
to the spatial arrangement of membrane proteins positioned
by the cytoskeleton that determines cell shape.[4]
After we had successfully proved our concept, we
explored the generality of the method: we performed
a series of experiments and found that the density of LHG
is variable and the ratio of 20nt and 6nt ssDNA can be as low
as 1:3, where the calculated coverage of the LHGs is less than
10% based on the relation A = 4pR2 (the radius of the AuNPs
is about 6.5 nm and the length of the DNA is about 6.8 nm).
We also found that this frame-guided assembly method could
be applied to different systems to obtain vesicles with
programmed geometry and dimensions, for example DNA-
PPO/amphiphilic block copolymers and cholesterol/sodium
dodecyl sulfate. Collectively, these results validate the adapt-
ability of our method.
In conclusion, we have proposed and demonstrated
a general assembly method: the frame-guided assembly, in
which LHGs are preanchored to the fringe of frame structures
and used to guide amphiphilic molecules to assemble into
heterovesicles. This method is modular and can be used to
assemble complexes of variable shape and size. We anticipate
that owing to the combination of DNA nanotechnology with
precisely defined addressable three-dimensional DNA nano-
structures utilized as a foundation,[11] our frame-guided
assembly method will enable the preparation of monodis-
persed vesicles, improve the understanding of the fundamen-
tal mechanism of self-assembly, and build up more complex
and functional assemblies beyond Au scaffolds.
Experimental Section
The AuNPs/AuNRs and the DNA-modified AuNPs/AuNRs were
synthesized following the procedure in previous reports.[12] The
synthesis of the DDPOG and G2Cl-18 is based on the solid-phase
method developed by our group.[7,8] The detailed description can be
found in the Supporting Information. A mixture of 30 nm DNA-
AuNPs and 5 mm DDOEG was incubated in 0.5 ꢀ TBE and 50 mm
NaCl at 48C for 3 h with a final volume of 10 mL. After the precipitate
had formed, the clear supernatant can be taken out directly. A 10 mL
aliquot of 5 mm G2Cl-18 was dissolved in 0.5 ꢀ TBE, and 50 mm NaCl
was added to the precipitate for about 10 min. After the mixture had
been shaken slightly, it was observed that the precipitate had
dissolved. UV/Vis spectroscopy and DLS were applied to character-
ize the changes in the sample.
Figure 3. DLS characterization. a) Unmodified 13nm AuNPs. b) DNA-
modified AuNPs with 20nt ssDNA. c) Heterovesicles with 20bp
dsDNA. d) Extended heterovesicles with 40bp dsDNA.
20nt and 6nt ssDNA to AuNPs at a ratio of 1:1, the radius of
the particles increased from 10.8 nm to 14.1 nm. 2) After the
formation of heterovesicles, the radius of the assemblies
further increased to 21.9 nm. This increase can be attributed
to the dsDNA, dendron layer, and the outer ssDNAs in the
final assemblies. 3) When the 20 base pair (bp) dsDNA was
replaced with a 40bp dsDNA, the radius increased to 29.4 nm.
This 7.5 nm increase matches well with the length of the 20bp
extension.[10] These results demonstrate that the size of
vesicles is determined by the frames, with parameters that
can be adjusted in the design, and the assemblies are egglike
vesicles. According to our strategy, the frame determines not
only the size but also the shape of the vesicle. This has been
proved by the following experiments: when the AuNPs are
changed to gold nanorods (AuNRs), the final assemblies are
rod-shaped vesicles (Figure 2d). This observation verifies that
the LHG molecules outlining the frame structure determine
Received: December 10, 2013
Published online: February 2, 2014
Keywords: frame-guided assembly ·
.
leading hydrophobic groups · self-assembly · vesicles
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