C O M M U N I C A T I O N S
Confocal laser scanning microscopy (CLSM) images collected at
Since we seek the production of vesicles with a single gp10 array
implanted in the membrane, low NTA-PEG600 surface coverages
are required for enabling adequate spacing between individual
surface-captured proteins in order to minimize vesicle-vesicle
encounters at the interface during connector array insertion into
the membrane. Increases in either the 3-aminopropyltriethoxysilane
concentration or the treatment time produced surfaces with very
large vesicles (>10 µm diameter) that were difficult to displace from
the surface, even at very high Im concentrations (CLSM data not
shown). We infer from these findings that high surface coverages
of gp10 connector protein lead to vesicle-vesicle fusion during
the surface-mediated reconstitution step. Since these very large
NTA-PEG-grafted glass surfaces revealed little or no nonspecific
2+
binding of the Ni :NTA-PEG surface (Figure 3A), but extensive
punctate surface staining was apparent on samples that had been
pretreated with N-his -gp10 connector arrays (Figure 3B). Adjustment
6
of the confocal plane to image the bulk phase revealed very little
rhodamine (Rh) fluorescence in solution due to the absence of unbound
vesicles. Addition of 2 M imidazole solution to these surfaces and
incubation for 10 min prior to CLSM imaging revealed the opposite
behavior, i.e., little or no surface fluorescence (Figure 3C) and extensive
punctate Rh fluorescence in the bulk solution (Figure 3D). TEM images
of the imidazole (Im)-desorbed vesicles showed the presence of
membrane-associated features whose size and shape are consistent with
gp10 connector arrays (Figure 4 and Figure S1). We infer from these
2
+
6
vesicles are presumed to possess numerous his :Ni :NTA surface
interactions, they would be expected to be difficult to competitively
6
observations that oriented insertion of N-his -gp10 connector proteins
occurred when they were exposed to small unilamellar C20BAS
bolalipid vesicles. This conclusion is supported by the following
observations: (1) absence of vesicle-associated fluorescence in samples
that were not exposed to connector protein (Figure 3A); (2) displace-
2
+
ment of vesicle-associated Rh fluorescence upon Im stripping of Ni
2+
from the his :Ni :NTA-PEG interaction (Figure 3D); and (3) highly
6
unfavorable interactions between the water-soluble PEG tether and the
hydrophobic core of the C20BAS membrane that would be expected
if the N-his terminus were oriented within the inner lumen of the
6
vesicle. Similar results were obtained when 7:3 C20BAS/Chol vesicles
were used in the surface-mediated reconstitution process. Taken
together, these findings suggest that the sequence depicted in Figure 2 is
a viable strategy for vectorial transplantation of N-his
arrays into host vesicle membranes of appropriate hydrophobic thickness.
6
-gp10 connector
Figure 4. CryoTEM images of N-his
6
-gp10 connector array-containing 9:1
C
20BAS/POPC vesicles produced as shown in Figure 2. The membrane-
2+
The reversible adsorption of 9:1 C20BAS/POPC vesicles from Ni :
NTA-PEG-grafted surfaces after transplantation of N-his gp10 connector
protein was also confirmed by atomic force microscopy (AFM). AFM
images collected after rinsing with Im solution revealed a surface with
few remaining vesicles (Figure S2). This result supports the conclusions
embedded gp10 arrays are highlighted with dotted circles.
6
In conclusion, Ni2+:NTA-PEG600-grafted glass surfaces are
capable of immobilizing N-his -gp10 from the phi29 DNA packag-
6
ing motor. C20BAS bolalipid vesicles stabilized by incorporation
of 10 mol % POPC or 30 mol % cholesterol can produce
reconstituted vesicles with transplanted connector arrays embedded
6
in the membrane upon exposure of the N-his gp10-primed surfaces
to sonicated C20BAS dispersions. These oriented gp10 vesicles were
detached by stripping the surface with excess Im, resulting in a
construct that is primed for subsequent pRNA and gp16 ATPase
binding to enable ATP-driven DNA packaging. Development of
this simple DNA packaging motor implantation strategy is an
important first step toward the practical realization of nonviral
vectors whose DNA cargo can be controllably encapsulated within
a carrier system having properties that can be readily optimized
for in vivo performance.
Acknowledgment. This work was supported by NIH (PN2 EY
0
18230 and CA112427) and KSF (KRF-2007-357-D00084). The
atomic coordinates for phi29 gp10 were obtained from the Protein
Data Bank under accession number 1FOU.
Supporting Information Available: Experimental methods and
TEM and AFM images. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
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Figure 3. CLSM images of Ni2+:NTA-PEG-modified glass surfaces after
exposure to 1% Rh-POPE-labeled 9:1 C20BAS/POPC vesicles. (A) Surface
image taken after protein-free surfaces were exposed to bolalipid vesicles
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(
3) Guo, Y. Y.; Blocker, F.; Xiao, F.; Guo, P. J. Nanosci. Nanotechnol. 2005,
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5
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7054 J. AM. CHEM. SOC. 9 VOL. 132, NO. 48, 2010
Hyun, Seok-Hee
