Precision templating with DNA of a virus-like particle with peptide nanostructures

Article abstract of DOI:10.1021/ja4008003

We report here the preparation of filamentous virus-like particles by the encapsulation of a linear or circular double-stranded DNA template with preassembled mushroom-shaped nanostructures having a positively charged domain. These nanostructures mimic the capsid proteins of natural filamentous viruses and are formed by self-assembly of coiled-coil peptides conjugated at opposite termini with cationic segments and poly(ethylene glycol) (PEG) chains. We found that a high molecular weight of PEG segments was critical for the formation of monodisperse and uniformly shaped filamentous complexes. It is proposed that electrostatic attachment of the nanostructures with sufficiently long PEG segments generates steric forces that increase the rigidity of the neutralized DNA template. This stiffening counterbalances the natural tendency of the DNA template to condense into toroids or buckle multiple times. The control achieved over both shape and dimensions of the particles offers a strategy to create one-dimensional supramolecular nanostructures of defined length containing nucleic acids.

Full text of DOI:10.1021/ja4008003

Article
pubs.acs.org/JACS
Precision Templating with DNA of a Virus-like Particle with Peptide
Nanostructures
Yves Ruff,† Tyson Moyer,‡ Christina J. Newcomb,‡ Borries Demeler,§ and Samuel I. Stupp*,†,‡,∥,⊥
‡
†Department of Chemistry, and Department of Materials Science and Engineering, Northwestern University, 2220 Campus Drive,
Evanston, Illinois 60208, United States
§Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC 7760, San
Antonio, Texas 78229-3901, United States
⊥
∥Department of Medicine, and Institute for BioNanotechnology in Medicine, Northwestern University, 303 East Superior Street,
Chicago, Illinois 60611, United States
S
* Supporting Information
ABSTRACT: We report here the preparation of filamentous
virus-like particles by the encapsulation of a linear or circular
double-stranded DNA template with preassembled mush-
room-shaped nanostructures having a positively charged
domain. These nanostructures mimic the capsid proteins of
natural filamentous viruses and are formed by self-assembly of
coiled-coil peptides conjugated at opposite termini with
cationic segments and poly(ethylene glycol) (PEG) chains.
We found that a high molecular weight of PEG segments was
critical for the formation of monodisperse and uniformly
shaped filamentous complexes. It is proposed that electrostatic attachment of the nanostructures with sufficiently long PEG
segments generates steric forces that increase the rigidity of the neutralized DNA template. This stiffening counterbalances the
natural tendency of the DNA template to condense into toroids or buckle multiple times. The control achieved over both shape
and dimensions of the particles offers a strategy to create one-dimensional supramolecular nanostructures of defined length
containing nucleic acids.
One of nature’s great examples of perfectly monodisperse 1D
assemblies is the tobacco mosaic virus (TMV), formed by
directed self-assembly of proteins (capsomers) on a RNA
template.10 Interestingly, TMV virions can be prepared in vitro
just by mixing the viral RNA genome and the purified capsid
proteins in aqueous solutions.11 Inspired by this process, we
described previously a templating strategy involving a dumb-
bell-shaped template and a self-assembling peptide amphi-
phile.12 This approach yielded nonspherical aggregates with
controlled dimensions measuring a few nanometers but was
impractical to create precise 1D assemblies with the length of a
filamentous virus particle. Synthetic analogs of viral capsid
proteins have been produced by genetic engineering or
chemical functionalization of the native protein to create
viruses as templates for the development of new materials or
bioactive nanostructures.13 Nucleic acids14 and negatively
charged polyelectrolytes15 have been used as templates to
reconstitute virus-like structures from native capsomers, but
control of the dimensions was only obtained in the case of
reconstituted closed icosahedral capsids. More recently, the
coating of single DNA molecules with genetically engineered
protein diblock copolymers was described,16 suggesting that the
INTRODUCTION
■
The field of molecular self-assembly has continued to evolve in
its capacity to design increasingly complex structures that are
not accessible by traditional covalent organic synthesis.1 One
important target that has been difficult is the one-dimensional
self-assembly of building blocks with precise length. The
growth of such assemblies occurs in an open mode and lacks
the precision of filamentous viruses, closed systems like
synthetic nanocages,2 or DNA-based nanostructures.3,4
A
number of bottom-up approaches have been proposed to
control the length of 1D self-assembled objects. For example,
one can control supramolecular polymerization by capping,5 or
the introduction of electrostatic repulsion between the
monomers to frustrate the growth of the assemblies,6 which
in both cases yield a broad statistical distribution of linear
supramolecular polymers. A better control can be achieved
using a vernier approach,7,8 yielding well-defined but low
molecular weight linear supramolecular aggregates. So far, low
polydispersity has been obtained only in organic solvents by the
careful control of nucleation and growth kinetics of
monodisperse cylindrical micelles formed by hydrophobic
crystalline block copolymers.9 Therefore, preparing homoge-
neous 1D nanostructures by molecular self-assembly remains a
great challenge, particularly in water.
Received: January 23, 2013
Published: April 10, 2013
© 2013 American Chemical Society
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Figure 1. Schematic representation of the strategy to prepare synthetic filamentous viruses using a DNA template and self-assembled mushroom-
shaped nanostructures created by the self-assembly of PEGylated coiled coil peptides modified by cationic spermine segments.
steric repulsion between the large colloidal stability domains
(400 amino acids) on the DNA-binding proteins contributes to
the stability of the complexes. To develop a system using easily
accessible synthetic building blocks, we attempted first to create
a monodisperse 1D assembly using a DNA template and simple
DNA-binding peptides, but observed instead uncontrolled
aggregation of molecules. We hypothesized that a preassembled
nanostructure analogous to a viral protein, with low enough
symmetry to contain a specific DNA-binding domain, might
yield a virus-like monodisperse structure and avoid the
uncontrolled aggregation we observed earlier. This Article
describes a successful strategy to create the virus-like particles
with defined length using as capsomers self-assembled
noncentrosymmetric nanostructures analogous to supramolec-
ular “mushrooms”.17,18 To create the mushroom nanostructure
as the artificial capsomer, we designed molecules able to bind a
nucleic acid 1D template. As depicted in Figure 1, the triblock
molecules contain a spermine unit at one terminus (Sp), a
known nucleic acid ligand,19 a coiled coil peptide (CC)20
known to aggregate into heptameric structures, and a long
poly(ethylene glycol) chain that provides solubility in water
(PEG2000 or PEG5000). We expected these molecules would
form uniformly sized capsomer-like aggregates due to the
synergy of a peptide predisposed to form finite heptameric
aggregates20 and entropy-limited aggregation mediated by the
long poly(ethylene glycol) segments.17,18 In the nanostructure
designed here, a third contribution to finite aggregation would
be the electrostatic repulsion among spermine segments.
CC-PEG2000 and Sp-CC-PEG5000 in phosphate buffer at pH
7.4 before and after addition of an excess of DNA (Figure 2A,
Supporting Information S6).21 For Sp-CC-PEG2000, the
proportion of ordered/disordered α-helix goes from 82:18
before to 100:0 after complexation with plasmid DNA, whereas
for molecule Sp-CC-PEG5000, this ratio is 75:25 before and
after addition of plasmid DNA. This slightly higher proportion
of disordered α-helix conformation could be explained by the
steric repulsion between the bulkier PEG5000 segments
clustered at the N-terminus of the self-assembled coiled-coil
peptides, as reported previously for PEG-coiled coil peptide
conjugates.22
We then investigated the coiled-coil formation further by
analytical ultracentrifugation, which gives access to the absolute
molecular weight of the aggregates in solution and subsequently
their aggregation number. Sedimentation coefficient (S20,w
)
distributions were calculated using the van Holde−Weischet
analysis.23 Sedimentation analysis from both equilibrium and
velocity experiments (Supporting Information S7)21 conducted
on Sp-CC-PEG2000 revealed the presence of a single species.
Specifically, for Sp-CC-PEG2000 at a concentration of 30 μM,
the van Holde−Weischet analysis suggested the presence of a
homogeneous solution. From further characterization of this
solute using the method known as two-dimensional spectrum
analysis and genetic algorithm−Monte Carlo (2DSA/GA-MC),
we determined a molecular weight of 33.74 kDa (95%
confidence intervals: 33.70, 33.78 kDa) and a weight-average
sedimentation coefficient of 2.28S (Figure 2B), indicating a
slight concentration-dependent nonideality (Supporting In-
formation S7).21 These molecular weights were confirmed by
equilibrium experiments that also suggest the presence of a
homogeneous 34.67 kDa oligomer (Supporting Information
S7)21 (RMSD = 4.64 × 10−03). These molecular weight values
are consistent with a hexameric assembly of Sp-CC-PEG2000
when we expected the GCN4-pAA peptide sequence to favor
RESULTS AND DISCUSSION
■
Characterization of the Artificial Capsomers. The self-
assembly of the triblock molecules Sp-CC-PEG2000 and Sp-CC-
PEG5000 into coiled-coil nanostructures was demonstrated
using circular dichroism and analytical centrifugation. Circular
dichroism (CD) shows >99% α-helical content for both Sp-
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○
▲
) at 30 μM and pH = 7.4 in phosphate buffer (25 mM,
Figure 2. (A) Circular dichroism of peptides Sp-CC-PEG2000 ( ) and Sp-CC-PEG5000
(
●
▲
150 mM NaCl). (B) Van Holde−Weischet analysis and integral distribution for Sp-CC-PEG2000 ( ) and Sp-CC-PEG5000
(
). The g(s)
distributions (solid line) obtained from a velocity experiment suggest the presence of a single, homogeneous solute. 2DSA/GA-MC analysis resulted
in a sedimentation coefficient of 2.28 s for Sp-CC-PEG2000 and 1.92 s for Sp-CC-PEG5000. (C) Agarose gel electrophoresis of complexes of Sp-CC-
PEG2000 and a 1200bp dsDNA fragment. Well 1, 200 ng of 1200 bp dsDNA fragment; wells 2−10, 200 ng of 1200bp dsDNA fragment+Sp-CC-
PEG2000 at concentrations of 5.6, 11.3, 17, 22.6, 33.9, 45.2, 56.5, 79, and 102 μM, respectively, using a total volume of 20 μL. (D) Agarose gel
electrophoresis of complexes of Sp-CC-PEG5000 and a 1200bp dsDNA fragment. Well 1, 200 ng of 1200bp dsDNA fragment; wells 2−10, 200 ng of
1200bp dsDNA fragment+Sp-CC-PEG5000 at concentrations of 3, 5.95, 8.9, 11.9, 17.8, 23.8, 29.75, 41.65, and 53 μM, respectively, total volume of
20 μL. (E) Agarose gel electrophoresis of complexes of Sp-CC-NH2 and a 300bp dsDNA fragment. Well 1, 200 ng of 300bp dsDNA fragment; wells
2−10, 200 ng of 300bp DNA fragment+Sp-CC-NH2 at concentrations of 2.5, 5, 7.5, 10, 15, 20, 25, 35, and 45 μM, respectively, using a total volume
of 20 μL.
the formation of a heptameric coiled-coil.20 However, as both
sedimentation velocity and sedimentation equilibrium experi-
ments only measure the buoyant molecular weight of the
sample, even a slight error (5%) in the estimate of the partial
specific volume (estimated at 0.81 mL/mg) could result in
fitting the data to a molecular weight consistent with a
heptameric aggregate (Supporting Information S7).21 There-
fore, our results are consistent with the formation of either a
hexameric or a heptameric coiled-coil aggregate, a behavior
consistent with the self-assembly of the GCN4-pAA sequence.
The van Holde−Weischet analysis for samples of Sp-CC-
PEG5000 at 15, 30, and 60 μM suggested the presence of
homogeneous solutions with a weight-average sedimentation
coefficient of 2.05, 1.81, and 1.98 at 15, 30, and 60 μM (see
Figure 2B for representative data). Monte Carlo analysis of the
velocity experiments (Supporting Information S7)21 on Sp-CC-
PEG5000 at these concentrations further confirmed that this
coiled-coil peptide forms oligomers, of which the major
components have an average degree of oligomerization
between 6.5 and 7.3 for an average molecular weight of 9229
g mol−1 for Sp-CC-PEG5000. Within the experimental error of
this experiment, these values are consistent with the formation
of heptameric coiled-coil aggregates.
In this assay, the reduction of the negative net charge of the
dsDNA template upon binding to the positively charged
peptides and the formation of higher molecular weight
complexes reduce mobility toward the anode (top) in the
agarose gel. After staining with ethidium bromide, we indeed
observed for Sp-CC-PEG2000 and Sp-CC-PEG5000 the
progressive retardation of the band associated with the
dsDNA/peptide complexes with respect to the free dsDNA
template. In both cases, a further increase in peptide
concentration led to a positive electrophoretic charge for the
complexes as they migrate toward the cathode. This charge
inversion indicates a complete neutralization of the negative
charges of the dsDNA template. In the case of Sp-CC-NH2, no
free DNA could be detected in the sample at a peptide
concentration of 10 μM corresponding to 200 peptide
molecules for one molecule of 1200bp dsDNA or 0.66 peptides
per base pair. This suggests that Sp-CC-NH2 is a better
condensing agent for dsDNA than Sp-CC-PEG2000 and Sp-CC-
PEG5000 requiring concentrations of 23 and 18 μM,
respectively, to completely neutralize one molecule of 1200bp
dsDNA (approximately 1400−1800 peptide molecules, which
corresponds to 1.2−1.5 peptides per base pair). A higher ratio
of peptide/dsDNA molecule could indicate either a lower
affinity for dsDNA or a denser coating of the bound dsDNA
molecules by the ligands. In addition, we showed that the
binding of Sp-CC-PEG2000 is not affected by physiological salt
concentrations (150 mM NaCl) by gel electrophoresis.21 In the
DNA Binding Studies. The ability of modified peptides Sp-
CC-PEG2000, Sp-CC-PEG5000, and Sp-CC-NH2 to bind nucleic
acids was demonstrated by agarose gel shift assays, with
different ratios of peptide to dsDNA template (Figure 2C−E).
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Figure 3. Effect of the PEG segment length on complex morphologies with circular plasmid DNA. (A) Complex between Sp-CC-NH2 and plasmid
pbr322, (B) complex between Sp-CC-PEG2000 and plasmid pbr322, and (C) complex between Sp-CC-PEG5000 and plasmid pbr322. (D) Relative
distribution of the three different complex morphologies observed, branched (B), toroidal (T), and rodlike (R), for (A), (B), and (C). (E)
Histogram length distribution of rodlike complexes between Sp-CC-PEG2000 and plasmid pbr322; this distribution is centered around Lth/2, which is
indicative of the folding of the DNA template. (F) Histogram length distribution of rodlike complexes between Sp-CC-PEG5000; this distribution is
centered around Lth, corresponding to the theoretical length of the extended supercoiled plasmid DNA template. For all samples, the plasmid pbr322
(200 ng) was added to the peptide at a concentration of 95 μM in 20 μL of 25 mM Tris buffer (pH 7.4, 25 mM). Scale bar = 200 nm for all images.
following TEM experiments, we ensured a complete neutraliza-
tion of the dsDNA templates by using a large excess of all three
ligands at a concentration of 95 μM.
morphologies (Figure 3B,D). This result is consistent with the
heterogeneous morphologies reported for PEGylated DNA-
polycation complexes24,29 and the phenomenon of DNA
condensation30 by multivalent cations, which is the transition
of DNA molecules from an extended coil to smaller ordered
particles, usually highly ordered toroids or rods containing
several DNA chains.30 Interestingly, we found that complexes
formed with Sp-CC-PEG5000 showed a dramatic improvement
in homogeneity in terms of both dimension and morphology.
Interestingly, for the complex pbr322/Sp-CC-PEG5000, we
observed only filamentous complexes with an average length of
about 727 nm (Figure 3C,D) and an extremely low
polydispersity index of 1.005. The length polydispersity index
(PDI) was calculated from the ratio of the number Ln and
weighted Lw length average by analogy to the standard
molecular weight polydispersity for polymers.31 The calculated
length of the pbr322 plasmid in a supercoiled conformation is
721 nm,21 within 1% of the observed length. This is a clear
indication that the circular dsDNA template is included in the
complexes in a tight supercoiled conformation, and that for
circular dsDNA, the theoretical length of the extended template
Lth should correspond to the length of the supercoiled
plasmid.21 We then prepared complexes of supercoiled
plasmids of different sizes using Sp-CC-PEG5000 as the ligand
(Figures 4 and Supporting Information S42). The analysis of
puC19/Sp-CC-PEG5000 and pKLAC1-malE/Sp-CC-PEG5000
again revealed homogeneous filamentous complexes. Further-
more, in these two additional cases, the length of the complexes
Structural Characterization of the Complexes: Effect
of the PEG Segment Length. Both conventional and
cryogenic microscopy as well as small-angle X-ray scattering
(SAXS)21 were utilized to image and characterize the
complexes formed between the preassembled nanostructures
and linear or circular dsDNA templates having different lengths
to probe the effect of the size and topology of the templates on
the morphology of the complexes. Figure 3 shows complexes
formed between a plasmid pbr322 DNA template and each of
the three molecules under conditions for which no free DNA
could be detected by gel electrophoresis. As shown in Figure
3A, an amorphous mass is observed when the plasmid is mixed
with positively charged molecules only containing the spermine
and peptide segments (Sp-CC-NH2). In the case of Sp-CC-
PEG2000, discrete complexes are observed, presumably
containing only one DNA template (Figure 3B,E). The
presence of a single DNA molecule is assumed based on
previous reports, indicating that PEGylation of DNA-polycation
complexes can prevent aggregation, and promote the formation
of complexes containing a single DNA molecule.24−29 While
discrete, the structures formed between the pbr322 plasmid and
Sp-CC-PEG2000 are heterogeneous and consist of rods,
branched, and toroidal complexes in a proportion of 36%,
23%, and 41%, respectively. Therefore, the majority of the
complexes formed in this case have either undefined or toroidal
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Figure 4. Effect of the size of the plasmid DNA template on the peptide/DNA complex length. (A) Complex between Sp-CC-PEG5000 and puC19
plasmid (2686 bp), (B) complex between Sp-CC-PEG5000 and pbr322 plasmid (4361 bp), (C) complex between Sp-CC-PEG5000 and pKLAC1-
malE plasmid (10153 bp), and (D) combined length distributions for complexes of Sp-CC-PEG5000 with puc19 plasmid (white bars; 2686 bp),
pbr322 plasmid (gray bars; 4361 bp), and pKLAC1-malE plasmid (black bars; 10153 bp). For all samples, the plasmid (200 ng) was added to the
peptide at a concentration of 95 μM in 20 μL of 25 mM Tris buffer (pH 7.4, 25 mM). Scale bar = 200 nm for all images.
essentially corresponds to the expected length of the super-
coiled plasmids. For complexes pKLAC1-malE/Sp-CC-
PEG5000, the average length of the complexes was 1619 nm,
very close to the calculated length of this plasmid in a
supercoiled conformation (1677 nm). These long complexes
were difficult to analyze, because the flexible filaments tend to
collapse on the carbon film of the TEM grid, making it difficult
to distinguish between branched structures and linear
complexes having overlapping segments. Therefore, for TEM
analysis of this sample, only complexes having an extended
supercoiled DNA template folded in half (Figure 3E). To better
understand the effect of the length of the template as well as the
influence of the ligand on this folding behavior, we conducted a
detailed study using linear dsDNA templates of different
lengths. This extension to linear dsDNA could also make our
approach applicable to a broader range of dsDNA templates,
which might be suitable to get smaller artificial viruses or to
encapsulate different kinds of nucleic acids such as siRNA.
Extension to Linear dsDNA and Discussion on the
Folding of the Complexes. Complexes between short linear
150bp dsDNA fragments and Sp-CC-PEG2000 (150bp/Sp-CC-
PEG2000) and Sp-CC-PEG5000 (150bp/Sp-CC-PEG5000) were
found to have a rodlike morphology and showed predom-
inantly the length of the DNA template (Figure 5A,B). The
dimensions of these complexes were determined to be 53 nm in
conformation were counted. For puC19/Sp-CC-PEG5000
pbr322/Sp-CC-PEG2000, and pKLAC1-malE/Sp-CC-PEG5000
,
,
toroid formation was also effectively suppressed, and the
filamentous morphology was the dominant morphology, which
is a dramatic improvement over literature precedents for
PEGylated cation/DNA complexes.24,29
We observed a low polydispersity index (1.011) for the
subpopulation of rodlike pbr322/Sp-CC-PEG2000 complexes,
even though the sample as a whole was heterogeneous. While
the expected theoretical length Lth of the supercoiled plasmid
pbr322 was about 721 nm, the observed distribution was
centered around 361 nm, which is the predicted length for a
length for 150bp/Sp-CC-PEG2000.
21 This result was confirmed
by SAXS experiments,21 which clearly showed the appearance
of rodlike complexes after mixing a 150 bp dsDNA template
with an excess of either peptide Sp-CC-PEG2000 or Sp-CC-
PEG5000. No folding or toroid formation was observed in this
case, because 150bp dsDNA fragments are known not to
undergo DNA condensation.32
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Figure 5. Effect of the length of the PEG segment on the peptide-linear double-stranded DNA complex morphology. (A) Complex between Sp-CC-
PEG2000 and 150 bp dsDNA fragment, (B) complex between Sp-CC-PEG5000 and 150 bp dsDNA fragment, (C) complex between Sp-CC-PEG2000
and 300 bp dsDNA fragment, and (D) complex between Sp-CC-PEG5000 and 300 bp dsDNA fragment. For all samples, the dsDNA (200 ng) was
added to the peptide at a concentration of 95 μM in 20 μL of 25 mM Tris buffer (pH 7.4, 25 mM).
Interestingly, in the case of 300bp/Sp-CC-PEG2000, the
length distribution obtained by TEM analysis was again
consistent with a folding mechanism for condensation, as
observed for pbr322/Sp-CC-PEG2000. Indeed, the observed
distribution was bimodal with maxima centered around the
expected length (Lth) as well as the length predicted for a DNA
template that is folded once (Lth/2) (Figure 5C). Thus, the
length distribution of rods is not random but corresponds to a
distribution of “quantized” lengths having a length L = Lth/(n +
1) of the plasmid DNA contour length, where n is the folding
number. Interestingly, for the 300bp/Sp-CC-PEG5000 com-
plexes, the population formed around the folded template is
smaller than the proportion of the same population for 300bp/
Sp-CC-PEG2000 complexes (Figure 5C,D). This implies that in
this case the forces opposing the folding are stronger when
nanostructures having a longer PEG segment are used to
complex the DNA. Because the only difference between Sp-
CC-PEG2000 and Sp-CC-PEG5000 is the N-terminal fragment,
we propose that the steric repulsion between PEG domains in
the preassembled mushroom aggregates results in an increase of
the persistence length of the complexes lp and prevents their
transition into shorter rods. This hypothesis was confirmed by
the length distribution of complexes between the more
sterically demanding ligand Sp-CC-PEG5000 and 600 or
1200bp dsDNA. We found that the maximum folding number
for 600bp/Sp-CC-PEG5000 complexes is n = 1, as compared to
n = 3 for 600bp/Sp-CC-PEG2000 complexes. Similarly, the
1200bp/Sp-CC-PEG5000 complexes showed a length distribu-
tion centered around the template folded once (n = 1, 204 nm).
We therefore conclude that the DNA binding nanostructures
formed by Sp-CC-PEG2000 and Sp-CC-PEG5000 interfere with
the folding process of the bound DNA template. These
observations are consistent with the quantized folding model
for DNA condensation proposed by Kataoka et al.24 In this
model, the discrete folded states in DNA−polycation
complexes originate from the Euler bending modes of initial
rodlike DNA−polycations complexes under a crushing force Fc.
For an internal DNA segment, the critical buckling force FEuler
was determined by Manning to be in the following form:33
B
FEuler = 4π2
L2
where B corresponds to the bending stiffness of the segment of
length L. The value for B is not known in our system, but it is
expected to depend on the persistence length lp of the
complexes, and therefore should be dependent on the ligand’s
structure, according to the following expression:33
lp = B/kbT
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Figure 6. Effect of the length of the PEG segment on the peptide-linear double-stranded DNA complex morphology. (A) Complex between Sp-CC-
PEG2000 and 600 bp dsDNA fragment, (B) complex between Sp-CC-PEG5000 and 600 bp dsDNA fragment, (C) complex between Sp-CC-PEG2000
and 1200 bp dsDNA fragment, and (D) complex between Sp-CC-PEG5000 and 1200 bp dsDNA fragment. For all samples, the dsDNA (200 ng) was
added to the peptide at a concentration of 95 μM in 20 μL of 25 mM Tris buffer (pH 7.4, 25 mM).
where kb is Boltzmann’s constant. As in the Euler buckling
model, increasing the folding number requires loads higher
than Fc to be applied to the initial rod structure,24 an increase of
lp, and the elastic bending stiffness of the rod B has a direct
effect on the extent of the folding.
Alternatively, another sequential folding mechanism has been
reported to explain the different stages of plasmid DNA
condensation by polylysine conjugates.34 In this case, increasing
the folding number would result in an increase in the charge
density of the folded DNA template, which in turn would have
to be compensated by an increase in the ligand density,
resulting in turn in increasingly stiffer rods. For sterically
demanding ligands, the optimum degree of folding might
therefore be determined by the balance between the steric
repulsion of the ligands and the electrostatic interactions with
the folded DNA template.
It is also important to note that the selectivity of the folding
process observed in the case of complexes with linear dsDNA is
consistent with the observations made for complexes with
supercoiled plasmid DNA. Indeed, due to the spatial proximity
between opposing plasmid segments, an extended supercoiled
plasmid DNA template can be considered as an analog to a
linear dsDNA template folded once,21 and having both of its
ends ligated. In addition, it has been shown that the screening
of the electrostatic repulsion between the juxtaposed super-
coiled DNA segments by divalent salts causes the transition
from a loosely to a tightly interwound superhelix to occur.35 As
an apparent complete charge neutralization of the DNA
backbone was observed by gel electrophoresis upon binding
of the supercoiled plasmid templates to our artificial capsomers
(Figure 2C−E), it is reasonable to expect the same transition to
occur in our system. In other words, supercoiling preorganizes
the circular DNA template and facilitates its transition into a
tightly interwound superhelix conformation equivalent to a
prefolded linear dsDNA template with a folding number n = 1.
Any subsequent folding of the supercoiled plasmid would yield
a template with a folding number equivalent to 3 or more for
linear dsDNA. In line with our observations, this implies that
complexes between a supercoiled plasmid DNA and Sp-CC-
PEG2000 would fold only once (Figure 3B,E), whereas
complexes with Sp-CC-PEG5000 should not fold because
folding numbers higher than one are not expected according
to the model established for linear dsDNA.
Increasing the bulk of the N-terminal fragments in Sp-CC-
PEG5000 nanostructures not only favored the formation of
essentially monodisperse filamentous nanostructures, but also
suppressed toroid formation. We found that when the length of
the template was increased above 300 bp, the morphology and
dimensions of the complexes between DNA fragments and Sp-
CC-PEG2000 became more heterogeneous as more toroids were
observed for 600bp/Sp-CC-PEG2000 and 1200bp/Sp-CC-
PEG2000 complexes (Figure 6A,C, Supporting Information
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Table S1). In contrast, complexes formed between linear
dsDNA fragments longer than 300bp and Sp-CC-PEG5000
showed consistently homogeneous rods having predominantly
a length corresponding to the DNA template folded once
without toroid formation (see Figure 6B,D, Supporting
Information Table S1). The morphology of the complexes is
also not affected by the presence of physiological salt
concentration,21 making our approach suitable for applications
requiring physiological buffers. Therefore, our approach can be
extended to the preparation of monodisperse filamentous
complexes from linear dsDNA templates. As previously
discussed, when supercoiled plasmids were complexed with
Sp-CC-PEG5000, homogeneous rodlike complexes were ob-
served as well, having in this case the length of the extended
supercoiled template.
NSF funded the synthetic work for the systems investigated.
Y.R. received partial support from a Lavoisier Fellowship from
the French Ministry of Foreign Affairs, and T.M. was supported
by a National Science Foundation Graduate Research Fellow-
ship. The authors thank the Biological Imaging Facility (BIF) at
Northwestern for the use of TEM equipment.
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■
The strategy described here allows precise control of the
dimensions of filamentous virus-like particles templated by
DNA. We showed that tuning steric forces among nanoscale
ligands binding to DNA templates makes it possible to obtain
homogeneous supramolecular rod-like objects either with
homogeneously folded dsDNA or extended supercoiled
plasmids reaching lengths of up to 1.6 μm. One-dimensional
supramolecular objects with precise length could have tunable
characteristics such as permeability, polyvalency, and extended
circulation times in biological systems, among others. At the
same time, they can carry precise cargoes of molecules or serve
as templates and building blocks for more complex structures.
Therefore, the principles learned here to create filamentous
supramolecular structures with precise length could be of
general interest in the design of future therapies and materials.
ASSOCIATED CONTENT
* Supporting Information
■
S
Materials and methods. Transmission electron microscopy
controls. Negative and positive staining of the complexes
between dsDNA templates and peptides Sp-CC-NH2, Sp-CC-
PEG2000, and Sp-CC-PEG5000. Effect of the peptides design on
the DNA toroids formation. Cryogenic transmission electron
microscopy. Circular dichroism. Analytical ultracentrifugation.
Small-angle X-ray scattering studies. Calculation of the
theoretical length of supercoiled plasmids. Additional TEM
images showing the effect of the size of the plasmid DNA
template on the peptide/DNA complex length. Stability of the
complexes to physiological salt concentration. Table for the
statistical analysis of the complexes. This material is available
free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
s-stupp@northwestern.edu
■
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the U.S. Department of Energy-
Office of Basic Energy Sciences, Division of Materials Sciences
and Engineering under Award No. (DE-FG02-00ER45810)
(Biomolecular Materials Program), and by the National Science
Foundation under Award No. (DMR-1006713). DOE funded
work on the characterization of self-assembled structures and
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