NANO LETTERS
A Virus-Based Nanoblock with Tunable Electrostatic Properties
2005 Vol. 5, No. 4 597-602
Anju Chatterji, Wendy F. Ochoa, Takafumi Ueno, Tianwei Lin,* and John E. Johnson* Department of Molecular Biology and Center for IntegratiVe and Molecular Biosciences, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California Received December 1, 2004
ABSTRACT Five different “HIS tag” mutants of cowpea mosaic virus were made by genetically introducing six contiguous histidine residues at various locations on the virus capsid. The mutant particles showed differential affinity for binding nickel, and their electrostatic properties could be controlled as a function of the protonation state of the exposed histidine sequence. The specific addressability of the HIS tag was corroborated by the selective modification of the histidine sequence with nanogold cross-linked to the Ni−NTA moiety.
The use of biomimetic peptides for attachment of metals to nanoparticles has become a method of choice for developing novel biomaterials for molecular electronics applications.1-8 Well-defined nanoparticles have been developed by the mineralization of cage-like proteins,9-15 but in recent years, the use of viruses as templates for mineralization has generated significant interest because of their potential as novel materials with optical, photonics or magnetic properties depending upon the kinds of metals that are packaged within the particles.10,16-19 Among various virus-based scaffolds that have been recently investigated for metallization purposes, cowpea mosaic virus (CPMV) offers a novel scaffold to serve as a protein platform primarily because extensive studies on the virus have established CPMV as an addressable nanoblock that can be specifically functionalized with a variety of ligands.20-23 The chemical and genetic modifications achieved on the exterior and interior interfaces of the capsid, while maintaining their stability and assembly, have allowed us to utilize the high symmetry of the viral cages to engineer unique functionalities for highly ordered multivalent presentation.24,25 In addition, the virus is exceptionally stable and can tolerate genetic insertions in several of its exposed loops, thus varying the accessibility of inserts. In this study, we explored the electrostatic behavior of CPMV/HIS mutants as a function of pH. Cowpea mosaic virus is a Picorna-like, icosahedral plant virus with a single-stranded, positive-sense RNA genome and is about 30 nm in size.26,27 Its capsid is formed by 60 copies of an asymmetric unit consisting of a small (S) and a large (L) subunit. The S subunit folds into one jelly roll β * Corresponding authors. E-mail:
[email protected] (T.L.); jackj@ scripps.edu (J.E.J.). 10.1021/nl048007s CCC: $30.25 Published on Web 03/01/2005
© 2005 American Chemical Society
sandwich domain, the A domain, and this clusters around a 5-fold axis. The L subunit is a single polypeptide chain that folds into two jelly roll β sandwich domains, B and C domains, that alternate around the 3-fold axis (Figure 1).28 Earlier work on CPMV involved creation and analysis of unique lysine and cysteine mutants of CPMV that were selectively modified to attach a variety of ligands on the virus capsid using the well-established amine or thiol chemistry.22 In this work, we have extended the capabilities of the virus scaffold by making five different viruses that present metal binding pockets on their surfaces. These were made by genetically modifying the viral RNA to express six contiguous histidine residues at four locations (two mutants are at the C-terminus) on surface-exposed loops on the capsid. This results in a charged local environment at specific sites along the virus surface that can be selectively functionalized to bind different metals that have an affinity for histidine residues. It is expected that the presence of a HIS-rich tag will allow pH dependent tunability of the virus electrostatic properties that may control chemical reactions conducted on this platform. There are several well-established insertion sites on the coat protein of CPMV that tolerate genetic modifications29 without compromising the infectivity of the virus (Figure 1). We chose to express the 6X HIS peptide at these sites to generate diversity in terms of the surface accessibility and conformation of the displayed sequence and on its behavior as an affinity tag using the well-established oligonucleotide directed mutagenesis techniques.30 Two (vaBC-H6 and vaCC-H6) of the five mutants have the HIS tag sequence displayed in the loops of the small subunit, while the vcEFH6 mutant has the 6X histidine peptide presented in the βE-
Figure 1. Space-filling model of CPMV capsid. The reference asymmetric unit is framed and the symmetry elements are labeled. The oval represents a 2-fold axis; the triangle is a 3-fold axis; and the pentagon a 5-fold axis. Locations of the six histidine inserts are shown relative to the virus particle (A); relative to the icosahedral asymmetric unit (B); and relative to the sequence # and β strands at which they were placed (C). The HIS tags on the virus particles and asymmetric unit are shown as a composite of all the inserts. In fact, each inserted sequence was placed individually on a single virus, creating five different virus mutants. Three of these mutants, vaBC-H6, (red), vaCC-H6 (green), and vcEF-H6 (teal), are presented in closed-loop conformation while the other two, vaCTr-H6 and vaCT-H6 (blue), are expressed as linear epitopes at the C-terminus of the small subunit. The prefix ‘v’ denotes the viral mutant and the ‘a’ or ‘c’ suffix indicates the corresponding domains of the small or the large subunit, respectively, that make up the asymmetric unit. The abbreviations and the colorcoding for different mutants are consistent throughout the text.
βF loop of the large subunit of the capsid. The other two mutants, vaCTr-H6 and vaCT-H6, are presented as linear epitopes on the C-terminus of the small subunit (Figure 1). The difference between these two mutants is dependent upon location of the displayed histidine tag at the C-terminus. The vaCTr-H6 mutants have a 23 amino acid truncation at their carboxyl end (last amino acid before the HIS tag is leu189) that occurs spontaneously during virus infection in plants, resulting in a faster migrating small subunit31 (21 kd instead of 23 Kd), while the full-length C-terminus mutants do not have the truncated C-terminus (the last amino acid being ala 213). All mutants developed systemic infections in plants. The yields of the mutants were similar to wild-type CPMV infected plants. An RT PCR assay with total RNA extracted 598
from the primary and the secondary leaves of the inoculated plants confirmed the presence of viral RNA transcripts (data not shown). To demonstrate that the HIS tag on various mutants was exposed and accessible, attempts were made to purify the virus from leaf homogenates using metal chelation chromatography. Traditionally, purification of virus from plant leaves is a tedious and time intensive process that requires repeated cycles of clarification in organic solvents and PEG precipitation followed by final purification on sucrose gradients by differential centrifugation to get rid of other plant proteins.32 For the HIS mutants, the clarified leaf homogenate was loaded directly on a Ni-NTA column and the purified virus was obtained using an immidazole gradient. Nano Lett., Vol. 5, No. 4, 2005
Figure 2. (A) Elution of various HIS mutants off of affinity column using an immidazole gradient. Differential affinity of the histidine mutants of CPMV for the Ni-NTA column was demonstrated by differences in the retention time of the mutants on the column when eluted with a 0-1 M immidazole gradient. The pH of the running buffer was adjusted to 7.0. (B) Immuno-blot analysis of the HIS mutants using anti-HIS antibody. The presence of the HIS tag on various mutants was confirmed by resolving the mutants on a 4-12% Bis-Tris gel and immunoblotting the proteins on a PDF membrane. The membrane was probed with anti-HIS antibodies. Of the five mutants, the vcEF-H6 mutant has the HIS peptide expressed in the large subunit of the CPMV capsid and was detectable as a 44 Kd band with anti-HIS antibody (lane 1), while the other four mutants expressing the histidine-rich peptide are present in the small subunit, which is visible as a 24 kd band (lanes 2-5) with anti-HIS antibody. (C) TEM analysis of the eluted virus particles from a nickel affinity column. The virus particles were stained with uranyl acetate (0.2%) and the images were obtained with a Philips Tecnai (100 Kv) electron microscope. The bar represents 100 nm. (D) Fluorescence emission spectra of the Newport Green dye derivatized samples excited at 510 nm. The relative degree of fluorescence is indicative of the differences in the amount of Ni2+ bound to the samples and reflected by the absorbance of the dye. (E) Quantitation of the nickel binding affinity of the HIS mutants. Relative numbers of dyes/particle bound to the mutants based on UV/vis spectroscopy. The fluorescence of the samples labeled with Newport Green dye was measured at 536 nm and calculated based upon the concentration of the virus particles.
The mutants did not bind the nickel affinity column equally well, suggesting their differential affinity for Ni2+. It is apparent from the chromatogram (Figure 2A) that the C-terminus mutants (vaCTr-H6 and vaCT-H6) had stronger affinity for the nickel column while the other (vaBC-H6, vaCC-H6 and vcEF-H6) mutants were retained on the column for a shorter period of time and eluted at relatively lower concentrations of immidazole. The eluted, purified virus fractions were analyzed on SDS PAGE using a 10% Bis Tris gel and tested in immunoblots using antihistidine antibody (Figure 2B). Examination of the eluted CPMV particles by TEM confirmed that they were intact, indicating that neither the presence of immidazole nor the purification process was detrimental to the virus particles (Figure 2C). The virus preparations were pure, but the yield of the mutants was low (0.1-0.2 mg/g of leaves) compared to traditional methods. As the goal of this study was to demonstrate that the introduced histidine-rich peptide sequence behaves similarly to the conventional HIS tag used to purify protein using the immobilized metal chelation affinity chromatography (IMCAC) method, the lower yields obtained with this method Nano Lett., Vol. 5, No. 4, 2005
of purification do not compromise the utility of these mutants. On the contrary, this tag offers a unique way to purify the virus for certain downstream applications that may either have to be performed rapidly, on a smaller scale or are incompatible with organic solvents. It should be noted that since some of these mutants (vaCTr-H6 and vaCT-H6) are presented as linear motifs while others in the loop (vaBCH6, vaCC-H6 and vcEF-H6) support a closed loop conformation, the location and relative accessibility of the HIS peptide are likely to influence the affinity of each HIS mutant for binding to the Ni-NTA resin. To get a measure of the relative binding affinities of each mutant for the Ni2+ ions, the virus preparations were incubated with metal ions (10 mM nickel sulfate solution) for an hour, followed by labeling with Newport Green dye that binds specifically to divalent metal ions. The excess dye was removed by successive rounds of passing the reaction mixture over a sephadex G50 column, and the final eluate was used for spectrophotometric analysis (see Supporting Information). The number of dye molecules bound to the virus was quantified by measuring the absorbance of the dye 599
Figure 3. Deprotonation of histidine-rich peptide at pH 9.0. The retention time of the native CPMV and the vcEF-H6 mutant is compared on the anion exchange column at pH 6.5 and 9.0. While the wt CPMV shows a small difference in terms of its retention time on the mono Q column at the different pH values (16.2 mL at pH 6.5 versus 17.5 mL at pH 9.0), the vcEF-H6 mutant shows a very dramatic shift from 12.2 to 17.6 mL, indicating that the histidine residues that were largely protonated at pH 6.5 are deprotonated at pH 9.0, thereby contributing no net charge on the virus surface. This behavior was verified by almost overlapping elution profiles of the wt and vcEF-H6 samples at pH 9.0.
at 510 nm. The dye quantitation experiments revealed that mutants vaCC-H6 and vaCTr-H6 promoted enhanced binding to the Ni2+ ions, while vaBC-H6, vcEF-H6, and vaCT-H6 displayed lower affinity for the metal ions (Figure 2D). As shown in the graph, (Figure 2E), the number of dye molecules conjugated to the virus particles varied with different mutants, ranging from 22 to 56 dye molecules per particle, confirming our earlier observations that not all mutants bind Ni2+ with equal affinity. The lower binding affinity of the vaCT-H6 mutant, even though its presented as a linear epitope, was related to spontaneous cleavage of the HIS peptide by the host/viral encoded protease, resulting in only a small fraction of the virus bearing the HIS peptide. Wild-type CPMV particles treated similarly with the Ni2+ and the Newport green dye did not show any measurable absorbance at 510 nm, indicating that they do not bind the Ni2+. Addition of EDTA to the labeled particles stripped off the fluorescence from the mutants, resulting in a dramatic loss of the absorption at 510 nm, confirming that the labeling was specific and targeted to HIS tags on the virus surface (Supporting Information). To demonstrate the influence of the HIS tag on the surface properties of viral capsids as a function of pH, the purified mutants were resolved on a mono Q anion exchange column. The wild-type CPMV has an isoelectric point of 5.533 and therefore at neutral pH binds to the anion exchange column. Changing the pH of the running buffer to 9.0 did not change the retention time of the native virus significantly, but a similar increase in the pH of the buffer was marked by a dramatic change in the binding affinity of the HIS mutants as the histidine residues are progressively deprotonated with an increase in the pH of the solution (Figure 3). The imidazole ring of the histidine residues is potentially protonated at slightly acidic pH values (pKa 6.7-7.1); therefore, at this pH range, these residues contribute to a net overall positive charge of the virus but at pH values greater than 600
the histidine pKa, the amines are not protonated and contribute no net charge to the virus, which explains the differences in the retention time of the histidine mutants at pH 6.5 (12.2 mL) and 9.0 (17.6 mL) on the mono Q column as compared to the wild-type CPMV (16.2 mL and 17.5 mL respectively; Figure 3). All the HIS mutants behaved in a similar fashion as demonstrated by their ion exchange profiles, with the change in the pH of the buffer. These results demonstrated that the surface properties of the histidine mutants could be modulated by adjusting the pH. The specific addressability of the mutants was verified by labeling the virus with nanogold derivatized with Ni-NTA cross-linker34 and therefore targets to only HIS sequences. We used cryo electron microscopy combined with threedimensional reconstruction techniques to determine the structure of gold-labeled vcEF-H6 mutant. Structural comparison between WT virus and the gold-labeled vcEF-H6 capsids revealed additional electron density located on the virus surface that can be attributed to the attached gold. To obtain the final 23 Å resolution map, a total of 1072 particles were selected.35 Due to the size of the molecule, gold particles were clearly identifiable under the microscope, allowing a selection of only those particles with strong density spots corresponding to gold molecules. A group of 900 particles was included in the new data sets for further processing.36,37 The different occupancy observed in the micrographs was resolved during the image reconstruction analyses due to the icosahedral averaging protocol.35 The final image shows a peculiar shape for the virus particle (Figure 4, left), where we can identify the wild type virus and additional density, mainly around the 5-fold axes (Figure 4, left). By subtracting the density for the wild-type particles from the density associated with the gold-labeled particles, the location of the gold is clearly defined38-40 (Figure 4, center). The density associated with the gold is extended on the particle surface just on top of the βE-βF loop where the HIS tag was inserted (Figure 4, right). The elongated density of the gold particles could result from either the flexibility of the histidine peptide or Ni2+ associated with the gold molecules binding to multiple histidine residues.33 In addition to the obvious utility of HIS mutants for nanochemistry and naomaterials applications, these mutants were used to confirm that the phenotype and the genotype were associated with the same particle in mixed infections of plants. This is a critical requirement for success in expressing combinatorial libraries of loops on the CPMV surface. To investigate the possibility of heterologous encapsidation of viruses when replicating in vivo, a mixture of wt CPMV and the vaCTr-H6 mutant of CPMV were coinoculated on young cowpea seedlings. The virus was purified from co-infected plants and analyzed by ion exchange chromatography. Earlier studies using individual virus samples had indicated that the retention volume of the wt CPMV and the vaCTr-H6 tagged CPMV varied with the pH of the buffer. While the wt CPMV invariably elutes at 36% B (16.5 min post-injection) at pH 6.5, the vaCTr-H6 mutant elutes at 30% B (10.5 min post-injection). The Nano Lett., Vol. 5, No. 4, 2005
Figure 4. (A) Cryo electron microscopy analysis of the vcEF-H6 mutant derivatized with Ni-NTA gold. (left) Three-dimensional reconstruction of the vcEF-H6 mutant conjugated with Ni-NTA gold at 23-Å resolution. The overall surface features of the particle appear different from the wt capsid surface (not shown). (center) Difference density (in yellow) was observed when the wt CPMV cryo EM reconstruction was subtracted from the gold labeled particles (shown on the left). The extra density corresponding to nanogold (gold) appears as islands/knobs protruding from the specific sites of insertion of HIS residues (6X HIS, residues 99-104). (right) Difference electron density, derived from vcEF-H6/Ni-NTA gold conjugate superimposed on the ribbon diagram of the asymmetric unit of the virus capsid. The histidine peptide is inserted in the βE-βF loop (teal) of the large subunit. The gold particle is drawn as a yellow sphere with a diameter of 14 Å. The gold is located approximately 30 Å from the βE-βF loop, which corresponds well with the 4-Å organic shell around the gold and approximately 10-Å length of the Ni-NTA cross-linker conjugated to the gold particles. The images were rendered with Molscript.36
Figure 5. Separation of a mixture of virus from plants co-infected with wt and vaCTr-H6 mutant. The individual viruses could be separated based upon the presence of a histidine-rich peptide on one of the samples using an anion exchange column. The retention time of the vaCTr-H6 mutant on a mono Q column at pH 6.5 is much lower (10.5 mL) than the native CPMV (16.5 mL) because the overall net positive charge on the particles is increased in case of the histidine mutant.
mixture of viruses purified from the co-infected plants was loaded on the ion exchange column and resolved at pH 6.5 using a 1 M salt gradient. Two virus peaks were observed (Figure 5) which corresponded to the retention time of the wt and the HIS mutant of CPMV. The virus peaks were collected and total RNA was extracted from the separated samples. RT PCR and DNA sequencing analysis of the fractions using HIS tagged virus primers confirmed the presence of the HIS epitope in the virus sample from the second peak, indicating that the coat protein of the histidine mutant encapsidated its own RNA genome. Similarly, the native RNA 2 sequence was obtained from the sequencing analysis of the peak 1 fractions. Taken together, these results indicate that there is a correspondence between the genotype and the phenotype of the CPMV particles during their multiplication and propagation in plants. Nano Lett., Vol. 5, No. 4, 2005
Recently, the use of bio-inspired nanomaterials has gained momentum for fabrication of a variety of nanodevices, primarily because of their versatility and their self-assembling properties, specifically using the histidine-rich peptides.41-44 CPMV is an excellent example of one such platform. In this work, we have added another dimension to this nanoblock by incorporating the histidine functionalities on the virus capsid, making it more amenable for metallization reactions. The histidine mutants investigated in this work incorporate all the advantages and utility associated with a traditional HIS tag for purification, screening, and detection purposes, while at the same time providing a simple approach to generate metal-virus composites either through covalent or via noncovalent attachment strategies. In addition, we have shown that the HIS tag provides tunability and control of the surface electrostatic properties of the capsid for chemical reactions as a function of pH. The creation of a collection of CPMV mutants with uniquely reactive HIS residues enriches the repertoire of addressable virus particles as nano building blocks. Acknowledgment. We thank Dr. George Lomonossoff for providing the infectious cDNA clones of CPMV. Funding from Office of Naval Research (N.00014-001-0671) to J.E.J. and N00014-03-1-0632 to T.L. is acknowledged. Supporting Information Available: Experimental procedures for generation and propagation of the mutants, nickel binding affinity of the mutants as a function of pH, and cryoelectron microscopy of the nanogold labeled histidine mutant are described. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Sarikaya, M.; Tamerler, C.; Jen, A. K.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2, 577-585. (2) Brown, S. Nat. Biotech. 1997, 15, 269-272. (3) Hilt, J. Z. AdV. Drug DeliV. 2004, 56, 1533-1536. 601
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NL048007S
Nano Lett., Vol. 5, No. 4, 2005
