Encapsulation of DNA-templated chromophore assemblies within virus protein nanotubes

Article abstract of DOI:10.1002/anie.201001702

Figure Presented A beneficial virus: The hierarchical self-assembly of a three-component system consisting of single-stranded DNA (oligothymines; Tq), chromophores (G), and virus coat proteins (CP) leads to the formation of micrometerlong nanotubes (see p

Full text of DOI:10.1002/anie.201001702

Angewandte
Chemie
DOI: 10.1002/anie.201001702
Self-Assembly
Encapsulation of DNA-Templated Chromophore Assemblies within
Virus Protein Nanotubes**
Andrꢀs de la Escosura,* Pim G. A. Janssen, Albertus P. H. J. Schenning, Roeland J. M. Nolte,
and Jeroen J. L. M. Cornelissen*
The use of biomolecules, which self-assemble to form precise
nanostructures, has attracted growing interest in nanotech-
nology over the last decade.[1–4] As an example, DNA has
been applied as a platform for the positioning of functional
molecules and nanoparticles with geometrical, size, and
positional control.[5–11] Viruses have also been used as robust
and monodisperse scaffolds for the incorporation of chemical
entities both in their inner cavities and on their exterior
surfaces.[12–17] Filamentous viruses such as the Tobacco mosaic
virus and M13 viruses can be used to construct one-dimen-
sional arrays, mainly through the attachment of small building
blocks to their surfaces.[18–21]
Herein, we describe a unique strategy to assemble
chromophoric molecules into very long helical stacks by
using both types of biological scaffolds outlined above, that is,
DNA to template the chromophore assembly[22–24] and the
Cowpea chlorotic mottle virus (CCMV) protein to create
nanotubes that can hold the DNA–chromophore architec-
tures.
CCMV[25,26] is an icosahedral virus with an outer diameter
of 28 nm and an inner cavity 18 nm in diameter. The capsid
shell is formed by 180 coat protein (CP) subunits that
assemble with T= 3 Caspar–Klug symmetry around a central
RNA matrix. The CP consists of 189 amino acids, with nine
basic residues at the N-terminal RNA binding domain. At
neutral pH and high ionic strength, CCMV virions disassem-
ble in vitro into protein dimers. After removal of the RNA,
CP dimers can be reassembled to form empty capsids, the size
of which varies with the assembly conditions. Furthermore, it
has been reported that when double-stranded DNA (dsDNA)
is encapsulated at neutral pH and low ionic strength, the CP
forms tubular structures 17 nm in diameter.[27]
We studied DNA-templated assemblies that consist of a
single-stranded DNA (ssDNA) template, that is, oligothy-
mines (Tq) of various lengths, to which naphthalene (G1),
stilbene (G2) and oligo(p-phenylenevinylene) (OPV; G3)
derivatives are bound by complementary hydrogen bonds. In
Tq–G complexes, one strand is DNA and the other strand is a
supramolecular stack of G molecules. Only one strand is
negatively charged, instead of two as for dsDNA. The DNA-
hybrid complexes normally adopt a right-handed helical
arrangement stabilized by p–p stacking and hydrophobic
interactions between the adjacent guest moieties, with the
phosphate groups pointing to the exterior of the backbone.
The interplay between the chromophore, DNA template, and
protein mantle results in a hierarchical organization of
components on different length scales, and the chromophores
within the tubes remaining in their helical arrangement
(Figure 1).
[*] Dr. A. de la Escosura,[+] Prof. R. J. M. Nolte,
Prof. J. J. L. M. Cornelissen[#]
Institute for Molecules and Materials
Radboud University Nijmegen
Toernooiveld 1, 6525 ED Nijmegen (The Netherlands)
Dr. P. G. A. Janssen, Dr. A. P. H. J. Schenning
Laboratory for Macromolecular and Organic Chemistry
Eindhoven University of Technology
PO Box 513, 5600 MB Eindhoven (The Netherlands)
[+] Current address: Organic Chemistry Department (Mꢀdulo 01)
We have studied the assembly of CP alone and in the
presence of T40, G1 and the T40–G1 complex. The concen-
tration of CP was 0.04 mm in all samples. All experiments
were carried out in 50 mm Tris-HCl buffer at pH 7.5 and low
salt concentration (0.1m NaCl). The temperature was kept at
58C, and 5 equivalents of G1 ([G1] = 1 mm) relative to the
thymine units ([T]T40 = 0.2 mm; [T]T40 represents the total
concentration of repeating thymine units in the assembly
sample) were used. These conditions ensure a high degree of
binding between G1 and T40 (Figure S1 in the Supporting
Information).[22] When the nonchiral diaminotriazine-func-
tionalized guest was added to Tq at low temperatures, an
induced bisignate Cotton effect was observed in the guest
absorption region (Figure S1a in the Supporting Informa-
tion). The chirality of the template is expressed in the
supramolecular organization of guest molecules, which shows
that they bind to Tq. These binding events result in a
rigidification of the ssDNA template, as was shown by cryo-
Universidad Autꢀnoma de Madrid
28049 Cantoblanco (Spain)
Fax: (+34)91-497-3966
http://www.orgchem.science.ru.nl/research/andresdelaescosura/
E-mail: andres.delaescosura@uam.es
[#] Current address: Laboratory for Biomolecular Nanotechnology
MESA+ Institute, University of Twente
PO Box 207, 7500 AE Enschede (The Netherlands)
Fax: (+31)24-36-53393
http://www.orgchem.science.ru.nl/people/jeroencornelissen.php
E-mail: j.j.l.m.Cornelissen@tnw.utwente.nl
[**] We acknowledge funding from the IEF Marie Curie program of the
European Union, the Chemical Council of the Netherlands Organ-
ization for Scientific Research (NWO), the EURYI Scheme of the
European Science Foundation (ESF), and the Royal Netherlands
Academy for Arts and Sciences. We also acknowledge Paul van der
Schoot and E. W. (Bert) Meijer for useful discussions, and Koen
Pieterse for the art work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201001702.
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Figure 1. Schematic representation of a) Tq–G complexes formed
between a single-stranded oligo(thymine) (Tq) (where q is the number
of thymine units, q=5, 10, 15, 20, 25, 30, 40) and guest molecules
equipped with a diaminotriazine functionality (G), and b) the assem-
blies formed by Tq–G in the presence of CCMV CP molecules. The
arrangement of the Tq–G templates within the tubes is a tentative
representation.
Figure 2. a) Circular dichroism spectrum of a mixture of T40, G1, and
CP in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl)
buffer (0.1m NaCl) at pH 7.5 and 58C ([G1]=1 mm, [T]T40 =0.2 mm,
and [CP]=0.04 mm). b) TEM micrograph taken from a mixture of T40,
G1, and CP under the same conditions (uranyl acetate staining).
c) Size distribution diagram of tube diameters in (b). d) TEM micro-
graph taken from a mixture of T40 and CP under the same conditions
(uranyl acetate staining). e) TEM micrograph showing the relative
sizes of the end cap of a tube and a T=1 capsid (uranyl acetate
staining). f) Photographs of the same lane in an agarose gel, which
contains T40, G1, and CP, visualized under a UV lamp (left) and by
coomassie blue staining (right).
TEM (Figure S1b in the Supporting Information) and molec-
ular dynamics simulations.[24]
When CP was mixed with a solution containing the T40–
G1 complex in a thymine/CP ratio of 10:1, the Cotton effect
of the complex was retained (Figure 2a), thus showing that
G1 maintains its helical arrangement. With TEM, under
uranyl acetate staining, many tubular structures of various
lengths and some capsids were observed (Figure 2b). All
capsids (ca. 10% of the total material under optimal con-
ditions; see below) had mostly the same diameter, corre-
sponding to T= 1 particles.[28] The tubes had a very uniform
diameter of (19 Æ 3) nm (Figure 2c) and hemispherical caps
similar in size as half of a T= 1 capsid (Figure 2d).
As a control experiment, the sample containing only CP
did not show any capsids or tubes when examined by TEM
(Figure S2 in the Supporting Information). The same result
was obtained when G1 was mixed with CP. For mixtures of CP
and T40 in a thymine/CP ratio of 10:1, T= 1 capsids were
mainly found on the TEM grids (Figure 2e).
The order of addition of the three components (i.e., CP,
T40, and G1) was found to dramatically influence the final
assembly product (see experimental procedures in the
Supporting Information). This result indicates a sequential
two-step self-assembly process, in which the formation of
T40–G1 occurs first and is followed by the nanotube assembly.
The influence of the Tq length on the self-assembly process
was also studied (Figure S3 in the Supporting Information).
Interestingly, tubes were formed even when complexes as
small as T5–G1 (ca. 2 nm in length) were used as templates.
Since the CP polymorphs were found to be stable upon
dilution,[29] they were also analyzed by gel electrophoresis in
agarose. Samples containing CP and T40–G1 gave an
elongated band eluting towards the anode, which was
attributed to the presence of tubes (Figure 2 f). Control
experiments were performed to ensure that this band was not
an artifact (Figure S4 in the Supporting Information). The
most remarkable observation from the gel electrophoresis
was that the eluting band that corresponded to the tubular
assemblies was clearly visible under UV illumination. There
also seemed to be accumulation of light-absorbing material
near the loading vessel, which may be due to very large tubes
that cannot migrate into the gel. These experiments con-
firmed the inclusion of ssDNA–guest complexes inside the
nanotubes.
In order to understand the role of T40 and G1 on the
polymorphism of the coat protein assemblies, titrations were
performed in which the relative concentrations of T40 and G1
with respect to the coat protein were systematically varied.
All the samples in a titration series were analyzed by TEM,
and the relative total areas occupied by tubes and capsids was
measured for each sample (Figure 3). These measurements
provided an estimate of the tendency of capsids and tubes to
be assembled in each case. Every sample was prepared
following the standard order of addition; that is, a solution of
CP was added to the T40–G1 complex.
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obtained for [G1]/[T]T40 = 1 than for higher ratios. For [G1]/
[T]T40 ꢀ 3, the length distribution of tubes did not change
dramatically, although a slight increase in the relative amount
of tubes longer than 1 mm could be observed with an
increasing [G1]/[T]T40 ratio.
In a second series of experiments, the CP concentration
was varied while keeping the T40–G1 concentration constant
(Figure S6 in the Supporting Information). In the absence of
CP, neither tubes nor capsids were obtained. An increase in
the amount of CP led to a significant increase of assembly
products. For [CP]/[T]T40 < 0.2, tubes were the main species
present (Figure 3c). At [CP]/[T]T40 = 0.1, the proportion of
tubular assemblies compared to capsids was at a maximum.
This ratio corresponds to 10 binding phosphate units per CP
subunit, and is remarkably close to the number of positively
charged residues in the N terminus of the coat protein
(9 residues).[25] At [CP]/[T]T40 ꢀ 0.2, tubes were still present,
but the fraction of capsids dramatically increased. The
average diameter of capsids also varied slightly with the
[CP]/[T]T40 ratio (Figure S7 in the Supporting Information).
Another important issue to be evaluated is the potential
of our procedure for assembling different p-conjugated
chromophores. To this end, two other diaminotriazine deriv-
atives G2 and G3 (Figure 1), which bear stilbene and oligo-
(p-phenylenevinylene) moieties, respectively, were synthe-
sized and tested for their capacity to bind to T40 and to induce
hierarchical assemblies with CP. Firstly, a concentrated
solution of G2 or G3 in DMSO ([G] = 0.04 mM) was injected
into a solution of T40 in Milli-Q water ([T]T40 = 0.02 mM).
Both samples showed an induced Cotton effect in the guest
absorption region, thus indicating that they bind to the T40
template. Remarkably, the Cotton effect of T40–G2 was
reversed, which suggests that the helicity of this complex may
be opposite to that of T40–G1 and T40–G3. The CP-
containing samples were prepared in the same way as
described above for experiments with G1, but for solubility
reasons these samples contained 2% DMSO, and the
concentrations of T40–G2 and T40–G3 were 25 times lower.
CD spectra showed that the Cotton effects were retained after
the samples were mixed with CP (Figure 4a), thus showing
that the helical structures of these T40–G complexes
remained intact. TEM studies revealed that tubes with
lengths over 2 mm had been formed (Figure 4b and Figur-
es S8,S9 in the Supporting Information) for both T40–G
complexes. The abundance of tubes on the grids was lower
than for the experiments with G1 because of the lower
concentration of the reagents. There were no tubes present in
the absence of the guest.
Figure 3. a) Plot of the ratio of total areas occupied by tubes and
capsids (measured by TEM) versus the [G1]/[T]T40 ratio, in a titration
where the concentrations of CP and template binding sites were kept
constant. [T]T40 =0.2 mm and [CP]=0.04 mm. b) Histogram showing
the length distribution of tubes for different [G1]/[T]T40 assembly ratios.
For [G1]/[T]T40 ꢁ3, an average of only ca. 30 tubes could be taken
because of the predominant presence of capsids. For [G1]/[T]T40 >3, an
average value of ca. 80 tubes is given. c) Plot of the ratio of total areas
occupied by tubes and capsids (measured by TEM) versus [CP]/[T]T40
in a titration where the concentration of T40–G1 was kept constant.
[G1]=1 mm and [T]T40 =0.2 mm. Each blue data point in graphs (a)
,
and (c) corresponds to the results from one micrograph, while the red
points represent average data points obtained from three different
micrographs.
A series of samples with variable amounts of G1 was first
prepared, while the concentrations of CP and T40 were kept
constant in a 5:1 ratio of CP and thymine units (Figure S5 in
the Supporting Information). When no guest was added, only
capsids were observed on the TEM grid. With each increase
of the concentration of G1, more fully bound T40–G1
complexes were formed and, as a consequence, the propor-
tion of tubes became larger (Figure 3a). The guest molecules
do not only exert an influence on the quantity of the
assembled nanotubes, but also on their lengths, as can be
concluded from the length distribution diagram in Figure 3b.
A much larger proportion of tubes shorter than 500 nm was
The mechanism of tube formation is very intriguing. The
assembly of CCMV tubular structures has been less explored
than the assembly of capsids. According to the tiling
theory,[30,31] CCMV tubes are thought to be rolled sheets
composed of protein subunits arranged in a hexagonal
lattice.[27] Very little is known, however, about the tube-
assembly process. Our results seem to show that the stiffness
of the polyanionic template is one of the main factors that
control which polymorphs are obtained at neutral pH. The
complexation of G molecules by Tq results in a rigidification
of the template, which forms a supramolecular double-
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[4] S. F. M. van Dongen, H. P. M. de Hoog, R. Peters, M. Nallani,
R. J. M. Nolte, J. C. M. van Hest, Chem. Rev. 2009, 109, 6212.
[5] K. Tanaka, A. Tengeiji, T. Kato, N. Toyama, M. Shionoya,
Science 2003, 299, 1212.
[6] N. C. Seeman, Nature 2003, 421, 427.
[7] B. Samorꢀ, G. Zuccheri, Angew. Chem. 2005, 117, 1190; Angew.
Chem. Int. Ed. 2005, 44, 1166.
[8] P. W. K. Rothemund, Nature 2006, 440, 297.
[9] T. H. LaBean, H. Y. Li, Nano Today 2007, 2, 26.
[10] O. I. Wilner, Y. Weizmann, R. Gill, O. Lioubashevski, R.
Freeman, I. Willner, Nat. Nanotechnol. 2009, 4, 249.
[11] M. Endo, H. Sugiyama, ChemBioChem 2009, 10, 2420.
[12] Q. Wang, T. W. Lin, L. Tang, J. E. Johnson, M. G. Finn, Angew.
Chem. 2002, 114, 477; Angew. Chem. Int. Ed. 2002, 41, 459.
[13] S. Sen Gupta, J. Kuzelka, P. Singh, W. G. Lewis, M. Manchester,
M. G. Finn, Bioconjugate Chem. 2005, 16, 1572.
[14] T. Douglas, M. Young, Science 2006, 312, 873.
[15] M. Uchida, M. T. Klem, M. Allen, P. Suci, M. Flenniken, E.
Gillitzer, Z. Varpness, L. O. Liepold, M. Young, T. Douglas, Adv.
Mater. 2007, 19, 1025.
[16] M. Fischlechner, E. Donath, Angew. Chem. 2007, 119, 3246;
Angew. Chem. Int. Ed. 2007, 46, 3184.
Figure 4. a) CD spectra of samples containing mixtures of T40, CP,
and either G2 (left) or G3 (right) as guests. b) TEM micrographs of
the tubular structures formed by G2 (left) and by G3 (right) in
combination with T40 and CP.
[17] A. de La Escosura, R. J. M. Nolte, J. J. L. M. Cornelissen,
J. Mater. Chem. 2009, 19, 2274.
[18] C. B. Mao, D. J. Solis, B. D. Reiss, S. T. Kottmann, R. Y. Sweeney,
A. Hayhurst, G. Georgiou, B. Iverson, A. M. Belcher, Science
2004, 303, 213.
[19] K. T. Nam, D. W. Kim, P. J. Yoo, C. Y. Chiang, N. Meethong, P. T.
Hammond, Y. M. Chiang, A. M. Belcher, Science 2006, 312, 885.
[20] Z. Niu, J. Liu, L. A. Lee, M. A. Bruckman, D. Zhao, G. Koley, Q.
Wang, Nano Lett. 2007, 7, 3729.
[21] Y. S. Nam, T. Shin, H. Park, A. P. Magyar, K. Choi, G. Fantner,
K. A. Nelson, A. M. Belcher, J. Am. Chem. Soc. 2010, 132, 1462.
[22] P. G. A. Janssen, S. Jabbari-Farouji, M. Surin, X. Vila, J. C.
Gielen, T. F. A. de Greef, M. R. J. Vos, P. H. H. Bomans,
N. A. J. M. Sommerdijk, P. C. M. Christianen, P. Leclere, R.
Lazzaroni, P. van der Schoot, E. W. Meijer, A. P. H. J. Schen-
ning, J. Am. Chem. Soc. 2009, 131, 1222.
[23] P. G. A. Janssen, J. L. J. van Dongen, E. W. Meijer, A. Schen-
ning, Chem. Eur. J. 2009, 15, 352.
[24] M. Surin, P. G. A. Janssen, R. Lazzaroni, P. Leclere, E. W.
Meijer, A. Schenning, Adv. Mater. 2009, 21, 1126.
[25] J. A. Speir, S. Munshi, G. J. Wang, T. S. Baker, J. E. Johnson,
Structure 1995, 3, 63.
[26] J. E. Johnson, J. A. Speir, J. Mol. Biol. 1997, 269, 665.
[27] S. Mukherjee, C. M. Pfeifer, J. M. Johnson, J. Liu, A. Zlotnick,
J. Am. Chem. Soc. 2006, 128, 2538.
stranded helix with all the negatively charged phosphate
groups pointing to the exterior of the backbone. Electrostatic
interactions between negative charges of the template and the
positively charged CP N terminus subsequently induce the
assembly of the coat proteins.
In conclusion, the hierarchical two-step self-assembly of a
three-component system, that is, guest, ssDNA, and CP, can
be instrumental in the encapsulation of stacking chromo-
phores over micrometer distances. We have formulated a
procedure to obtain virus protein nanotubes where any
organic chromophore bearing a diaminotriazine unit could
potentially be encapsulated. It is noteworthy that the tubular
lengths exceed that of the DNA template by two orders of
magnitude. Our studies open unique possibilities to arrange
functional molecules into robust nanostructures with con-
trolled geometry, size and position.
Received: March 22, 2010
Keywords: DNA structures · nanostructures · nanotubes ·
.
[28] T= 1 capsids are formed by 60 coat protein subunits.
[29] The Cotton effect was retained even under tenfold dilution,
under which conditions G1 normally does not bind to T40, as
shown by the absence of a CD effect if no CP is present.
[30] R. Twarock, Bull. Math. Biol. 2005, 67, 973.
self-assembly · viruses
[1] C. M. Niemeyer, Angew. Chem. 2001, 113, 4254; Angew. Chem.
Int. Ed. 2001, 40, 4128.
[31] T. Keef, A. Taormina, R. Twarock, J. Phys. Condens. Matter 2006,
18, S375.
[2] V. M. Rotello, J. Mater. Chem. 2008, 18, 3739.
[3] A. J. Dirks, R. J. M. Nolte, J. J. L. M. Cornelissen, Adv. Mater.
2008, 20, 3953.
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