Fabrication of Massive Sheets of Single
Layer Patterned Arrays Using Lipid
Directed Reengineered Phi29 Motor
Feng Xiao,†Jinchuan Sun,‡Oana Coban,†Peter Schoen,§Joseph Che-Yen Wang,?R. Holland Cheng,?and
nology.1?5Most of the current micro- and
nanofabrication approaches use a variety of
robust materials to generate morphologies
with typical geometrical shapes such as
chip, pillars, bar, or pyramids. Superlattices
have been fabricated by various physical or
chemical methods including deposition,6?8
phy,25or patterned etch pits.26The con-
struction of lattices that mimic the struc-
tural complexity of biological structures
would be very intriguing and challenging.
In nanotechnology, a nanomachine is a
mechanical or electromechanical device
with nanometer size dimensions.27,28Con-
siderable efforts have been focused on the
he ability to synthesize patterned
arrays in a controllable fashion is of
extensive interest for nanotech-
research and development of nanoma-
chines and their potential applications in
medical related fields. One promising av-
enue is to construct synthetic nanoma-
chines that mimic the powerful natural bio-
nanomachines and to incorporate them
into traditional nanotechnological
applications.29,30Living systems manufac-
ture a large variety of nanomachines made
of protein,31DNA,32and RNA33with atomic
precision, including motors, arrays, pumps,
membrane cores, and valves. The structural
and conformational complexity of biologi-
cal molecules brings about new avenues
and challenges to experimental approaches
at the bio/nano interface.1,4,33?36For in-
stance, fabrication of replica-molded biom-
aterials requires precise topographical pat-
terning. Two-dimensional crystals of
proteins have been reported growing on a
lipid monolayer at an air?water interface
by using Langmuir deposition or by spread-
ing a protein solution at the liquid?air
interface.1,37,38Efficient and reproducible
transfer of such 2D crystalline protein films
onto solid substrates would have substan-
tial implications in the design of nanotech-
nological devices. Similarly, protein adsorp-
tion on solid substrates with predictable
self-assembly patterns is another valuable
tool for nanopatterning. The use of
building blocks in nanotechnology has an
advantage over the chemical materials in
their eligibility for site-directed modification
and specific conjugation with defined stoi-
chiometry. In addition, they can self-
assemble and be connected directly to bio-
*Address correspondence to
Received for review June 29, 2008
and accepted November 24, 2008.
Published online December 16, 2008.
10.1021/nn800409a CCC: $40.75
© 2009 American Chemical Society
nanomimetics. In this paper, we report on the assembly of single layer sheets of reengineered phi29 motor
KEYWORDS: bacteriophage phi29 · portal vertex · dodecamer · single layer
VOL. 3 ▪ NO. 1 ▪ XIAO ET AL. www.acsnano.org
logical molecules. Self-assembly
of molecules on a surface can be
a simple, versatile, and high-
volume production approach for
the construction of biological
arrays.5,40?44The ability to repli-
cate biological shapes with
nanoscale precision could have
profound implications in tissue
engineering, cell scaffolding,
drug delivery, sensors, imaging,
One particularly attractive
candidate found in the viral
DNA-packaging machinery, from
which both protein and RNA bio-
nanocomponents may be har-
vested, is the Bacillus subtilis bac-
teriophage phi29 DNA-
powerful motor comprises a por-
tal vertexOa 12-subunit gp10
(dodecamer protein, also called
and ATP55?57Othat provides
the chemical energy required
for DNA packaging. These com-
ponents can be combined in
powerful nanomachines con-
structed to date.58,59
The class of dodecamer (con-
nector) proteins which form vari-
eties of portal vertex shares little
sequence homology among dif-
ferent viruses, but the resulted
portal vertex has considerable
Information from Cryo-EM64and
portal vertex is a 12-fold sym-
metric dodecamer with a truncated cone shape about
7.5 nm long and with a diameter of 6.8 nm at the nar-
row end (N-terminus) and 13.8 nm at the wide end (C-
terminus). The central channel has a diameter of 3.6 nm.
The wide end of the dodecamer is embedded in the
procapsid shell, while the narrow end of the
hold for pRNA binding.66,67
Previous work has shown that the wild-type
dodecamer can be used to form arrays with a mix-
ture of single, double, or multiple layered
structures.47,68Multilayer arrays could be easily pro-
duced from ordered aggregates of portal vertex.
However, multilayer arrays are of limited use for
nanotechnological applications such as replica,
which demand uniform, single layer biomolecular ar-
rays. It has been found that some multilayer crys-
tals can be converted to single layer in solution over
a period of a few weeks by gradually changing the
salt concentration.68However, such a step is time-
consuming and only generates small sheets as a mix-
ture with single, double, and mutlilayers. Due to
the thinness and frangibility, purification of the
single layer from the mixture is almost impossible.
In this study, we demonstrate that we can easily
produce huge two-dimensional single layer arrays
of terminus-modified portal vertex using a lipid
Figure 1. Multilayer versus single layer sheet arrays of phi29 motor dodecamer. (A) Side view of a
multiple layer dodecamer array showing the horizontal face-up and face-down arrangements and
the vertical head-to-tail alignment which leads to multiple layers overlap. (B) Side view of a single
layer dodecamer array displaying an alternating face-up and face-down arrangement. (C) Native
phi29 motor dodecamer (inset) assembled into ordered multiple layer structures as shown by
negative-stain electron micrograph. (D) The negative-stain electron micrograph of reengineered
phi29 motor dodecamer (inset) arrays shows that a single layer sheet was formed. (E) Projection
density map of the single layer of motor dodecamers and the Fourier transform (inset). The unit cell
is rectangular, with a lattice constant of ?20 nm. The alternate orientations of the dodecamer can
be observed. (F) AFM image of N-strep dodecamer arrays and a line scan across crystalline area with
lattice defects (inset). The height difference between the top dodecamer layer and mica surface is
?7.5 nm, which corresponds to a single dodecamer layer.
www.acsnano.orgVOL. 3 ▪ NO. 1 ▪ 100–107 ▪ 2009
monolayer as template. The method is simple and
RESULTS AND DISCUSSION
Single Layer Patterned Arrays. The formation of multilayer
arrays is driven by two distinct protein interaction
mechanisms. First, horizontal side-by-side interactions
between individual dodecamers allow for the extension
tions between the narrow and the wide ends of
dodecamer molecules promote the buildup of mul-
tiple layers vertically (Figure 1A). To facilitate the forma-
tion of a single layer and prevent the continuous
growth of multiple layers, a short peptide sequence
was introduced either into the gp10 N- or C-terminus,
located at the narrow and wide end of the dodecamer,
respectively (Figure 1B).
vertex is a truncated cone-shaped structure having the
gp10 N- and C-terminus located at the narrow and wide
end, respectively. Fusion of a simple 22 amino acid
Strep-tag to the N-terminus of the portal vertex did
not interfere with the assembly of the quaternary
dodecamer structure. After expression in E. coli cells,
the recombinant gp10 assembled into dodecamer par-
ticles with similar shape to the native portal vertex as
shown by TEM (data not shown). Additionally, the
Strep-tag extension facilitates purification of the
dodecamer protein with high yield and homogeneity.
It has been previously reported that two-dimensional
dodecamer arrays could be grown in solution from con-
centrated native dodecamer (connector) solution fol-
lowing several weeks of incubation under a defined
ionic strength gradient of the buffer.47,68However,
without this precise chemical treatment, the native por-
tal vertex has the tendency to form patches of mul-
tiple layers. Figure 1A illustrates a multiple layer struc-
ture of native dodecamers. The individual dodecamers
interactions. We have used a reengineered motor
dodecamer for the self-assembly of single layer
dodecamer sheets (schematically shown in Figure 1B).
Arrays constructed from both native (Figure 1C inset)
and reengineered dodecamer (Figure 1D inset) and im-
aged by TEM are shown in Figure 1C,D. As previously re-
ported, the unmodified dodecamer generated mul-
tiple overlapping layers with tetragonal symmetry
(Figure 1C). The different shades of gray represent the
different layers formed and overlapping. The extent to
which different layers overlap cannot be controlled, and
thus it is difficult to reproduce the same multilayer
structure. Interestingly, the reengineered dodecamer
with the added N-terminal extension self-assembled
into huge flat sheets that piled into three-dimensional
stacks (Figure 1D). Such stacks are different from 3D
crystals, of which the stacks are governed by specific in-
teraction between different layers. However, in these
stacks, the sheets arrange in random orientation (Fig-
ure 2), suggesting that each sheet formed or grew inde-
pendently. It is understandable that, without a sup-
port, the fragile sheet of the thin layer could not stand
alone (see next section for formation of single layer
sheets on supporting lipid monolayer). From the EM im-
ages, it appeared that the size of the center channel
was smaller (compare Figure 1C and D). This is possi-
bly due to partial filling-up of the edge of the channel
by the extended peptide at the N-terminus.
The arrangement of the individual dodecamers was
analyzed using statistical classification and averaging
(Figure 1E inset). The truncated cone structure of a
single dodecamer enables us to distinguish between
the face-up and face-down orientations. The corre-
sponding projection density map shows the alternat-
ing face-up and face-down arrangement of dodecam-
of ?20 nm. Due to the alternating orientations of the
motor, the central face-up dodecamer displays a larger
diameter (corresponding to the wider C-terminal end)
each corner of the square. The alternative face-up and
-down data agree with the previous studies on EM im-
aging, X-ray crystallography, topological analysis, struc-
ture projection of the 2D crystals, 3D reconstruction,
and computer modeling of the dodecamers.47,64,65,69
tion of single layer arrays was further confirmed by
AFM imaging. Freshly cleaved muscovite mica was used
as an alternative substrate. Figure 1F shows a typical
Figure 2. Negative-stain electron micrographs and corresponding fast Fourier transform (FFT) of two-layer patterned sheets
of N-strep dodecamer. (A) Self-assembled huge flat sheets piled into 3D stacks. (B) Representative fast Fourier transforma-
tion. (C) The red and blue circles in the image suggested two layers sheets slightly arranged in different angle, suggesting
that each sheet formed or grew independently and stacked together.
VOL. 3 ▪ NO. 1 ▪ XIAO ET AL.www.acsnano.org
AFM image of crystalline N-strep dodecamer single lay-
ers imaged in tapping mode in liquid. Patches of crys-
talline areas with submicrometer size can be observed.
The large scan image shows a high surface coverage of
the protein layer with only small imperfections. A line
scan across the single layer sample (Figure 1F inset) in-
dicates that the single layer thickness is ?7.5 nm, which
is in excellent agreement with the height of the motor
dodecamer determined from the three-dimensional
crystal structures.53Similar single layer arrays were also
produced from dodecamers of gp10 with a peptide ex-
tension at the C-terminus which serves as a barrier for
the vertical interactions (data not shown).
Monolayers. Nanotechnological applications of arrays re-
quire the assembly of homogeneous broad and wide-
ranging flat sheets. Due to the flexible and fragile na-
ture of proteins, it would be desirable to employ a
biological template to direct the assembly of single
layer arrays. To explore this possibility, thin layers of
biotinylated lipid mixtures were used to direct the as-
sembly of N-strep dodecamers which carry a Strep-tag
at the N-terminus of each gp10 subunit. The N-strep
dodecamer was preincubated with streptavidin before
the lipids were spread. Arrays were grown in situ on
lipid monolayers at the air?water interface and then
transferred on carbon-coated TEM grids. Saturated di-
palmitoyl fatty acid chains (C16) of biotinyl dap DPPE
were mixed with unsaturated phosphatidylcholine fatty
acids chains of egg PC (C18) in a 1:3 (w/w) ratio and
spread at the liquid?air interface. Dodecamers were at-
tached to the lipid surface via specific
biotin?streptavidin interactions (Figure 3A). Initially,
the N-strep -tagged dodecamer bound to streptavidin
is randomly oriented in solution. After the lipid mixture
containing the biotinylated lipid is applied on the wa-
ter surface, the N-strep dodecamer/streptavidin com-
plex binds specifically to the biotinylated lipid. The ar-
rangement of the individual dodecamers is dictated by
the intermolecular protein contacts and protein?
surface interactions. The distance between subsequent
dodecamers is governed by the intrinsic nature of the
protein which exhibits strong side-by-side interactions.
Protein?protein, protein?lipid, and lipid?lipid interac-
tions contribute to the single layer arrangement
dodecamers in the lipid matrix. The unsaturated dilut-
ing lipid egg PC provides fluidity and flexibility to the
lipid monolayer. The bound protein is carried by the bi-
otinylated lipid through the lipid matrix which confers
to the translational and rotational freedom required for
the nucleation and growth of crystalline patches. After
a hydrophobic grid and imaged by TEM (Figure 3B).
The Fourier transform and the corresponding projec-
tion density map of the negatively stained electron mi-
crograph (Figure 3C and D, respectively) revealed tet-
ragonally packed 2D dodecamer crystals with a unit cell
size of ?18 ? 18 nm2. The unit cell dimensions are in
close agreement with those previously measured on
two-dimensional phi29 dodecamer crystals by TEM.68
philic bare mica was used as alternative surface for the
assembly and adsorption of single layers of the reengi-
neered dodecamer with either N- and C-strep modified
dodecamers. High-resolution images of the patterned
surface are shown in Figure 4. The N-strep dodecamers
self-assembled into a parallelogram lattice (Figure 4A).
Cross-sections along the x (Figure 4B) and y (Figure 4C)
directions of the crystalline areas indicated unit cell di-
mensions of ?16 and ?13 nm in the x and y directions,
Figure 3. Lipid-directed formation of single layer dodecamer arrays. (A) Schematic illustration of experimental approaches. (1)
Streptavidin/N-strep dodecamer solution is placed in a Teflon well; (2) biotinylated lipids DPPE and helper lipids egg PC were spread at
the air?water interface to attract dodecamers to the surface of the liquid; (3) dodecamers bound to the lipid via specific
biotin?streptavidin interactions. (B) Negative-stain TEM image of a single layer array produced with the aid of a lipid monolayer. (C) Fou-
rier transforms and (D) corresponding Fourier projection maps of lipid-directed N-strep dodecamer arrays.
www.acsnano.orgVOL. 3 ▪ NO. 1 ▪ 100–107 ▪ 2009
respectively. The angle between the x and y axis has
been calculated to be ?71°. Even though the tetrago-
nal arrangement previously observed was maintained,
the slightly different and unequal unit cell dimensions
suggest that the packaging unit of this type of crystal
might be slightly different from that of the lipid-
directed N-strep 2D crystal. The crystal lattice in Figure
4 is different from those in Figures 1?3 concerning the
angle of the pattern. While asking whether the differ-
ence observed in the lattices was the consequence di-
rectly related to the mutation of the protein is very in-
triguing, still little is known. A rectangular lattice with
unit cell dimensions of 18 ? 18 nm2has been observed
for the C-strep mutant. The dodecamer orientation in
the self-assembled layer on the mica surface was simi-
lar to that in the three-dimensional crystal and gener-
ated face-up and face-down arrangements. While occa-
sionally the low force applied for imaging was sufficient
to image what appeared to be the narrow ends of the
dodecamer, most of the time we could only visualize
the wide dodecamer domains due to the nature of
tip?sample interactions in AFM imaging.
Mica has more than 10 different phases concerning
the surface lattice. The lattices of mica and dodecamer
are of a different order of magnitude, in which mica is
calculated about 6 Å compared to the protein with a
unit cell in the regime of about 16–18 nm. In this AFM
imaging, it is not clear whether the mica surface lattice
played a role here in organizing the pattern of the pro-
teins array, and whether the mica surface lattice and the
protein crystal lattice are relevant or in a good match.
However, previous studies have shown that the purified
native dodecamer self-assembled into tetragonal ar-
rays in solution without the mica support,47guiding of
these nanoparticles by the mica surface lattice to form
the pattern in this report might not be necessary. In-
stead, the pattern of the lattice might have been dic-
tated by the intrinsic property of the mutant
A short Strep-tag sequence modification of the N-
or C-terminus of the phi29 portal vertex facilitates its
purification with high yield and homogeneity. The
modification did not interfere with the dodecamer as-
sembly and function. The mutant protein exhibited fa-
vorable lateral interactions and led to the formation of
large dodecamer sheets. In solution, the 2D dodecamer
arrays interacted vertically to pile up into 3D stacks of
protein sheets as revealed by TEM imaging. Large single
layer sheets of highly ordered array have been con-
structed using a supporting lipid monolayer.
tex protein were engineered by attaching a Strep-tag II (WSH-
PQFER) to either the N-terminus or the C-terminus of each gp10
subunit. Cloning methods of the N-strep and C-strep dodecamer
have been described previously.70
with 1 mL of Strep-Tactin sepharose resin (IBA, St. Louis, MO)
was equilibrated with 10 column bed volumes of buffer W (100
ter lysis of E. coli cells containing the reengineered gp10, the ly-
sate was clarified and the supernatant was loaded onto the col-
umn, followed by washing with buffer W, the protein was eluted
with buffer E (500 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin,
100 mM Tris-HCl, pH 8.0, 15% glycerol).
Assembly of Dodecamer Arrays. Two approaches were used to
construct dodecamer arrays: (1) self-assembly from concen-
trated solutions of purified native and N-strep or C-Strep motor
dodecamer and (2) lipid-directed assembly of single layer
dodecamer arrays. The schematic illustrations of multilayer ar-
rays or single layer patterned sheets are shown in Figure 1A,B.
Self-Assembled Dodecamer Arrays. Concentrated solutions of puri-
fied, reengineered C- and N-strep dodecamer in buffer (100
mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA, 0.02% sodium azide
15% glycerol, pH 8.0) were stored at ?20 °C. Protein solu-
tions dialyzed and diluted if necessary to a stock of 1 mg/mL
were kept at 4 °C for a few days and used for the construc-
tion of two-dimensional arrays. A 1:35 dilution of the protein
stock solution in imaging buffer (10 mM Tris-HCl, pH 8.0,
500 mM KCl) was applied on freshly cleaved mica. The sample
was placed in a humidified, closed Petri dish to avoid drying
out. Following 2 h incubation at room temperature, the
sample was rinsed with imaging buffer and kept at 4 °C over-
night. The sample was allowed to reach room temperature
prior to AFM imaging.
dodecamer arrays were grown at the liquid?lipid interface as
previously reported by Sun et al.71,72The N-strep dodecamer at
a concentration of 0.1 mg/mL was incubated in buffer (50 mM
Figure 4. High magnification AFM images of self-assembled arrays of reengineered motor dodecamers. (A) Tetragonal ar-
rays of N-strep dodecamer. (B and C) Cross-sections along the axes of the two-dimensional array. The unit cell is a parallelo-
gram with cell dimensions of 16 nm ? 13 nm. (D) Tetragonal arrays of C-strep dodecamer. The unit cell is rectangular with
a lattice constant of ?18 nm.
VOL. 3 ▪ NO. 1 ▪ XIAO ET AL. www.acsnano.org
Tris-HCl, 100 mM NaCl, 20 mM MgCl2, pH 8) at room tempera-
ture with a 6-fold excess of streptavidin (Sigma). A volume of 15
?L of the N-strep dodecamer bound to streptavidin was placed
into a custom-designed Teflon well of 4 mm in width and 1 mm
in depth. The lipid mixture of 30 ?g/mL biotin-cap-DPPE and
90 ?g/mL egg phosphatidylcholine (Avanti Lipids, AL) was pre-
pared in chloroform, and 0.3 ?L of the lipid mixture was layered
on top of the protein solution and incubated overnight at 4 °C
in a humidified chamber.
(TEM). Samples were prepared by applying the protein stock
solution on hydrophilic glow discharged carbon grids that
were negatively stained with 1% uranyl acetate (UA) or 2%
(w/v) ammonium molybdate. The two-dimensional arrays
grown on the lipid matrix were transferred to a hydropho-
bic carbon-coated copper grid without glow-discharged,
washed with distilled water, and negatively stained with an
aqueous solution of 1% UA and imaged by TEM. The images
were acquired at 45000? magnifications on a Philips CM12
TEM operated at 80 kV acceleration voltages and equipped
with a CCD camera (Gatan, Inc., PA). The reengineered phi29
motor dodecamer arrays were applied on glow-discharged
carbon-coated grids, washed with distilled water, and nega-
tively stained 2% ammonium molybdate. The grids were
transferred into a JEOL-2100F TEM operated at 120 kV, and
the images were acquired at 40000? magnification on a 4k
? 4k CCD camera (TVIPS, Germany). Image processing, struc-
tural determination, and three-dimensional reconstruction
(3D) were carried out by the electron crystallographic
method using CRISP software package.73The two-
dimensional (2D) projection map of the dodecamer array
was generated using the EMAN software as described else-
to maintain the sample under buffer at all times. Samples were
allowed to reach room temperature before being imaged by
AFM. The self-assembled dodecamer arrays were imaged in liq-
uid in tapping mode using a Nanoscope III multimode instru-
ment (Veeco/Digital Instruments, Santa Barbara, California)
equipped with a 130 ?m scanner (J scanner). Tapping in liquid
was performed in a buffer droplet in a tapping-mode liquid cell
without an O-ring seal. Scanning was performed using narrow-
legged cantilevers (OMCL-TR400PSA, Olympus Ltd., Tokyo, Ja-
pan) with oxide sharpened Si3N4tips. The V-shaped cantilevers
had a length of 100 ?m and a nominal spring constant of 80 pN/
nm. Cantilevers were driven at the resonance frequencies of 8.4
? 0.5 kHz with piezo drive amplitudes of 50?100 mV, resulting
in cantilever amplitudes of ?0.5 V. Scanning was performed at
ing) and data analysis was done with the Nanoscope software
Acknowledgment. We thank D. Green from University of Cali-
fornia at Davis for his technical assistance and valuable com-
ments, as well as the preparation of Figures 1D and 2. This work
was supported by PN2 EY018230 from NIH Nanomedicine Devel-
opment Center for Phi29 DNA Packaging Motor for Nanomedi-
cine through NIH Roadmap for Medical Research.
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