Langmuir 2007, 23, 2663-2667
2663
TMV Microarrays: Hybridization-Based Assembly of DNA-Programmed Viral Nanotemplates Hyunmin Yi,†,‡ Gary W. Rubloff,†,§ and James N. Culver*,| Department of Materials Science and Engineering and Institute for Systems Research, UniVersity of Maryland, College Park, Maryland 20742, and Center for Biosystems Research, UniVersity of Maryland Biotechnology Institute, College Park, Maryland 20742 ReceiVed August 23, 2006. In Final Form: December 4, 2006 The intrinsic ability of biological molecules to self-organize into complex structures has the potential to revolutionize methods for the assembly of nanomaterials and devices. In this work, nucleic acid hybridization was used to simultaneously assemble different Tobacco mosaic virus (TMV) nanotemplates onto a glass substrate patterned with address specific capture DNAs. To accomplish this, TMV-based nanotemplates were programmed with linker DNAs containing sequence specific addresses and hybridized directly to the capture DNAs. This assembly process proved to be a reliable, selective, and controllable means to assemble multiple TMV nanotemplates.
Introduction The self-assembly properties of biologically derived macromolecules offer distinct advantages for engineering novel materials and devices at a nanoscale level. For example, assembled virus particles have been genetically and chemically engineered as scaffolds for the display of functional groups including metals, dyes, and antigens.1-5 Functionalized viruses have also been tested as microreactors, battery electrodes, and digital memory devices.1,6,7 However, the assembly of most biologically derived nanotemplates has generally been limited to the random deposition of functionalized particles onto a solid substrate or device with little or no ability to control their spatial orientation. The random nature of this deposition process and lack of spatial control represents a significant source of inefficiency and limits the use of these templates in the construction of nanodevices. Thus, new methodologies that permit the targeted assembly of biologically derived nanotemplates to patterned substrates are needed. Previously, we demonstrated the ability to position Tobacco mosaic virus (TMV) nanotemplates onto gold-patterned silicon substrates using nucleic acid hybridization.4 TMV has an 18 nm × 300 nm rod-shaped particle composed of ∼2130 identical coat protein subunits (MW 17.5 kDa7) encapsulating a single strand of plus sense genomic RNA. Additionally, TMV has * Corresponding author. Tel.: (301) 405-2912. Fax: (301) 314-9075. E-mail:
[email protected]. † Department of Materials Science and Engineering, University of Maryland. ‡ Current address: Department of Chemical and Biological Engineering, Tufts University, Medford, MA 02155. § Institute for Systems Research, University of Maryland. | University of Maryland Biotechnology Institute. (1) Douglas, T.; Young, M. Nature 1998, 393, 152-155. (2) Mao, C. B.; Flynn, C. E.; Hayhurst, A.; Sweeney, R.; Qi, J. F.; Georgiou, G.; Iverson, B.; Belcher, A. M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (12), 6946-6951. (3) Shenton, W.; Douglas, T.; Young, M.; Stubbs, G.; Mann, S. AdV. Mater. 1999, 11 (3), 253-256. (4) Yi, H. M.; Nisar, S.; Lee, S. Y.; Powers, M. A.; Bentley, W. E.; Payne, G. F.; Ghodssi, R.; Rubloff, G. W.; Harris, M. T.; Culver, J. N. Nano Lett. 2005, 5 (10), 1931-1936. (5) Haynes, J. R.; Cunningham, J.; Vonseefried, A.; Lennick, M.; Garvin, R. T.; Shen, S. H. Biotechnology 1986, 4 (7), 637-641. (6) Nam, K. T.; Kim, D. W.; Yoo, P. J.; Chiang, C. Y.; Meethong, N.; Hammond, P. T.; Chiang, Y. M.; Belcher, A. M. Science 2006, 312 (5775), 885-888. (7) Tseng, R. J.; Tsai, C.; Ma, L.; Ouyang, J.; Ozkan, C. S.; Yang, Y. Nat. Nanotechnol. 2006, 1, 72-77.
evolved a defined disassembly mechanism that initiates with the removal of coat protein subunits from the 5′-end of the viral RNA.8 This initiation process occurs upon cell entry but can also be mimicked in vitro by alkaline treatment. We took advantage of this disassembly process to hybridize the exposed TMV 5′genome sequence to complementary probe DNAs attached to a chitosan-patterned gold surface. This process permitted both the spatial and the orientational alignment of virus derived nanotemplates onto a solid surface. In this study, we demonstrate that differentially labeled TMV nanotemplates can be simultaneously addressed to defined positions on a solid surface. As shown in Scheme 1, genetically modified TMV nanotemplates are first labeled with fluorescent markers and partially disassembled by alkaline treatment. This treatment selectively removes ∼20 coat protein subunits from the virus 5′-end, exposing ∼60 nucleotides of the viral RNA. Partially disassembled nanotemplates are then programmed with linker DNAs that contain sequences complementary to both the virus 5′-end and a selected capture DNA probe. Programmed TMVs carrying linker sequences readily assembled onto corresponding capture DNAs printed on a patterned microarray platform. Results demonstrated that high spatial and sequence specificity are obtained during nanotemplate hybridization, including density control through the modulation of capture DNA concentration. These findings demonstrate that programmed nucleic acid hybridization provides a facile and highly controlled process for the assembly of multiple TMV-based nanotemplates onto generic solid platforms. Materials and Methods TMV Purification and Fluorescent Labeling. Infectious RNA transcripts were generated from a full-length TMV1cys cDNA construct as previously described9,10 and used to inoculate Nicotiana tabacum, cv Xanthi. Inoculated plants were harvested after 20 days and virus purified as described.11 Purified TMV1cys (400 µg) was incubated for 1 h with a 5-fold molar excess of Cy5 or Cy3 maleimide (8) Namba, K.; Pattanayek, R.; Stubbs, G. J. Mol. Biol. 1989, 208 (2), 307325. (9) Dawson, W. O.; Beck, D. L.; Knorr, D. A.; Grantham, G. L. Proc. Natl. Acad. Sci. U.S.A. 1986, 83 (6), 1832-1836. (10) Turpen, T. H.; Reinl, S. J.; Charoenvit, Y.; Hoffman, S. L.; Fallarme, V.; Grill, L. K. Biotechnology 1995, 13 (1), 53-57. (11) Gooding, G. V.; Hebert, T. T. Phytopathology 1967, 57 (11), 1285.
10.1021/la062493c CCC: $37.00 © 2007 American Chemical Society Published on Web 01/30/2007
2664 Langmuir, Vol. 23, No. 5, 2007
Yi et al.
Scheme 1. Hybridization-Based Programming of Fluorescently Labeled and Partially Disassembled TMV Nanotemplates for Assembly onto Multiple Addresses on DNA Oligonucleotide Microarray Platforms
Figure 1. Sucrose gradient centrifugation of Cy5- and Cy3-labeled TMV nanotemplates. Unreacted dyes remain at the top, while labeled TMVs appear in the middle as distinctive bands.
Figure 2. Patterned assembly of fluorescently labeled and programmed TMV nanotemplates onto oligonucleotide microarrays via hybridization of Cy5-TMV1cys-D and Cy3-TMV1cys-H simultaneously. (a) Alternating pattern of sequences D and H (Table 1). (b) Alternating pattern of sequences G and H.
(Amersham Biosciences, Piscataway, NJ) in 50 mM Tris-buffer, pH 7.0. Cy5- or Cy3-labeled virus was separated by centrifugation in a 10-40% sucrose gradient at 56 000g for 2 h, and the pH was adjusted to 8.0 to partially remove coat protein subunits from the 5′-ends of the viral genome. Partially disassembled virions were pelleted by centrifugation for 30 min at 66 000g. Pelleted viruses (10-20 µg/mL) were resuspended in 4 × SSC buffer (30 mM sodium citrate, 300 mM sodium chloride, pH 7.0). Preparation of Spotted Capture DNA Microarrays. HPLCpurified single stranded capture DNAs with primary amine group end modification (Table 1) were purchased from Gene Probe Technologies (Gaithersburg, MD) and used without further purification. These solutions were printed on aldehyde functionalized glass slides (SuperChip aldehyde enhanced surface, Erie Scientific Co., Portsmouth, NH) by an Affimetrix 417 arrayer (Santa Clara, CA) in array mode with a 300 µm spot-to-spot distance according to the manufacturer’s protocol. Briefly, upon printing, the slides were
incubated in the arrayer overnight at room temperature and then thoroughly rinsed with distilled water. The slides were then incubated in dilute sodium borohydride solution (3:1 mixture of phosphate buffered saline (PBS) and ethanol) for 20 min with 75 rpm gyratory shaking to stabilize the Schiff base linkage and then thoroughly rinsed with distilled water and dried under nitrogen gas. Assembly of TMV Nanotemplates via Hybridization. For address specific programming of labeled and partially disassembled TMV, molar excess of linker DNAs D and H (Table 1) were added to Cy5-TMV and Cy3-TMV solutions, respectively, and incubated at 30 °C for 2 h. To remove unbound linker DNAs, mixtures were centrifuged at 66 000g for 30 min in 5 × SSC buffer. The pellets were resuspended in 5 × SSC buffer, and the two TMV solutions were mixed equally and incubated with the capture DNA microarrays. Microarrays were hybridized overnight at 37 °C and then sequentially washed with 50 mL of 5×, 4×, and 2× SSC at 37 °C for 20 min each followed by PBS and distilled water washes at room temperature. Washed microarrays were dried under nitrogen gas before visualizing fluorescence. Analysis. The microarrays were scanned with a GenePix Professional 4200A scanner (Molecular Devices, Union City, CA)
Assembly of DNA-Programmed Viral Nanotemplates
Langmuir, Vol. 23, No. 5, 2007 2665
Figure 4. Efficiency of the hybridization-based programming for addressable TMV nanotemplates. The region within the white rectangle has 72 capture DNA spots complementary to either the TMV 5′-end or the TMV 3′-end genomic sequences. Red spots on the right side of the panel are derived from spotted capture DNA sequence D used as a positive control to confirm successful printing by pin #4 (Supporting Information). The hybridization mixture contained Cy5-TMV1cys-D and Cy3-TMV1cys-H.
Figure 3. Assembly density control of TMV nanotemplates via capture DNA concentration. Hybridization mixture contained Cy5TMV1cys-D (red) and Cy3-TMV1cys-H (green). (a and b) Patterned capture DNAs D and H, respectively, at concentrations of 16, 32, 63, 125, 250, and 500 µg/mL. (c) Fluorescence intensity plot averaged from patterned Cy5-labeled (red) and Cy3-labeled (green) TMV nanotemplates shown in panels a and b. for Cy3 and Cy5 fluorescence. The fluorescence images were then analyzed with ScanAlyze software (http://rana.lbl.gov/EisenSoftware.htm) for the fluorescence intensity of each spot (Figure 3c and Supporting Information). Average fluorescence intensities from the 12 spots per column were plotted with ( one standard deviation (Figure 3c). Scanning electron microscopy (SEM) images were obtained as described previously.4 Briefly, the microarray slides were cut into 8 mm × 25 mm pieces and sputter coated with 60:40 platinum/palladium alloy and viewed using a Hitachi S.4700 FE-SEM (Pleasanton, CA).
Results and Discussion Differential Assembly of TMV Nanotemplates. TMV1cys encodes a cysteine residue at the amino terminus of the viral coat protein.4 This genetic alteration produces surface exposed thiol groups along the entire outer surface of the virus rod. To visualize differentially addressed virus templates, thiol-reactive Cy5- and Cy3-maleimide-linked dyes were attached to the TMV1cys surface. Gradient centrifugation confirmed coupling of the fluorescent markers and was used to purify labeled virions away from the unreacted dye, as shown in Figure 1.
The distinctive bands for both Cy5- and Cy3-labeled TMV shown in Figure 1 along with previous work demonstrating the attachment of metal clusters4 confirms the generic template capabilities of TMV1cys using standard thiol-reactive chemistry. We took advantage of TMV’s natural disassembly process to expose the 5′-end of the virus genome while leaving the bulk of the virus particle intact. Exposed 5′-end genome sequences can provide a sequence specific tag for the patterned assembly of functionalized viruses.4 However, the exposed virus genome sequence provides only a single address for use in template patterning. In this study, we demonstrate that the exposed virus 5′-end could be modified, or programmed via hybridization with a linker DNA, to impart multiple sequence specific addresses, thus greatly expanding the ability to pattern TMV templates (Scheme 1). By design, these linker DNAs can contain any sequence but must include a region complementary to the virus 5′-end, as shown in Table 1. To validate our ability to differentially address TMV nanotemplates via hybridization, we programmed by hybridization specific linker DNAs onto Cy5- and Cy3-labeled TMVs. Programmed TMV templates were then hybridized to a DNA microarray platform, containing spotted single stranded capture DNAs of various sequences and concentrations. As shown in Figure 2a, one region of the microarray was patterned with alternating spots of D and H capture DNA (Table 1). Microarray hybridization with TMV templates programmed with D (Cy5-labeled, shown as red) or H (Cy3-labeled, shown as green) produced a clear alternating and bright red-green fluorescence pattern that corresponded with the spotted capture DNAs. Another region on the same microarray was patterned with alternating spots of the H capture DNA and a nontargeted
2666 Langmuir, Vol. 23, No. 5, 2007
Yi et al.
Table 1. Single Stranded DNA Sequences and 5′-End/3′-End Modificationsa
a Asterisk denotes FITC: fluorescein isothiocyanate labeled onto the capture DNA with sequence D for preinspection of the DNA printing procedure via a fluorescence microscope (Supporting Information).
G capture DNA, as shown in Figure 2b. TMV hybridization in this region produced consistent fluorescence (green spots) at the targeted H sequence spots but no detectable fluorescence at the nontargeted G sequence spots. Combined, these results demonstrate that programmed TMV templates can be correctly assembled onto a solid substrate with both high spatial resolution and little if any cross-hybridization or background fluorescence. Importantly, this simple hybridization-based programming strategy for multiple nanotemplate assembly offers several unique advantages. First, it eliminates the need for arduous genetic manipulations or chemical procedures to impart multiple address specific information onto the virus template. Second, the highly selective nature of nucleic acid hybridization allows template programming to be simple, specific, and reproducible, even for a large number of addresses. Third, hybridization through the virus 5′-end imparts orientational control over the attachment of the virus template, resulting in less aggregation and more uniform arrays of patterned templates. Modulation of Assembly Density by Capture DNA Concentration. Altering the capture DNA concentration used for microarray printing provided a simple method to control nanotemplate assembly densities (Figure 3). Specifically, spotted capture DNA solutions of varying concentrations, ranging from 16 to 500 µg/mL for sequences D (Figure 3a) and H (Figure 3b), were spotted onto the microarray platform. Simultaneous hybridization studies using D (red) and H (green) programmed TMVs showed increases in template derived fluorescence that correlated well with increasing concentrations of the spotted capture DNAs, as shown in the fluorescence intensity profile of Figure 3c. Additionally, there was little or no cross-hybridization between programmed TMVs and capture DNAs as demonstrated by the absence of yellow spots throughout the various capture DNA concentrations. These results indicate that nanotemplate assembly densities can be easily controlled through the concentration of the capture DNA. Efficiency of Hybridization-Based Programming. To test the efficiency of linker DNA programming, capture DNAs complementary to the 5′- and 3′-end sequences of TMV genomic RNA were patterned onto the microarray (white rectangle shown
in Figure 4) and exposed to Cy5- and Cy3-labeled TMVs programmed with D and H linker sequences, respectively. Results show no detectable hybridization of D or H programmed TMVs to either the 5′- or the 3′-end specific capture DNAs. These findings indicate that exposed TMV 5′-end sequences are fully occupied by linker DNAs and that the disassembly process does not result in the exposure of 3′-end viral sequences. Note that the right side in Figure 4 shows red fluorescence from spots containing capture sequence D used as a positive control to confirm successful printing of the specific area as well as to pre-inspect the printed microarrays by fluorescence microscopy. Additionally, pretreatment of the labeled and partially disassembled TMV with RNase before prehybridization with linker DNAs resulted in no fluorescence upon microarray assembly, confirming the hybridization-based nature of both the programming and the assembly (data not shown). Furthermore, linker hybridized TMV templates could be stored for several days at 4 °C without a notable loss in hybridization efficiency (data not shown). Thus, the addition of address specific DNA linkers represents an efficient and robust method for the production of address specific viral nanotemplates. Scanning Electron Microscopy (SEM) of Assembled TMV Nanotemplates. To directly visualize nanotemplate assembly density and integrity, we examined microarray-assembled TMV templates via SEM, as shown in Figure 5. As evident in the electron micrographs, TMV nanotemplates were assembled with high density and spatial selectivity. Figure 5a shows an SEM image of one of the TMV-assembled spots, with a diameter of roughly 16 µm, which is easily recognized due to the high density of the TMV template. Figure 5b,c shows close-up views of the boundary between the TMV-assembled templates and the glass platform, showing both high spatial resolution and virus density. Additionally, the overall integrity of assembled TMVs was wellmaintained throughout the extensive rinsing, drying, and high vacuum conditions necessary for SEM sample preparation, demonstrating the stability of these hybridized patterned templates. Further, the SEM images correlate well with the high fluorescence signals and extremely low backgrounds shown in
Assembly of DNA-Programmed Viral Nanotemplates
Langmuir, Vol. 23, No. 5, 2007 2667
clearly illustrate the efficiency of the hybridization-based assembly of TMV nanotemplates onto solid surfaces.
Conclusion Biologically derived materials, such as TMV, hold the potential for enhancing or imparting new functionalities to devices at the molecular level. However, the need to integrate such biologicals into more traditional top-down fabricated nanoscale devices represents a significant obstacle in their use. One technology that has been the focus of intense research over the past decade involves the integration of DNA into fabricated devices for use in sensors and genetic assays. In addition to such ample options available for simple surface modification with DNA, the hybridization-based assembly strategy offers several unique advantages such as unsurpassed selectivity, practically unlimited number of addresses, and mild aqueous processing/assembly conditions without arduous and/or special chemical procedures or drying. In this paper, we have taken advantage of existing DNA immobilization technologies to direct the patterning of TMV nanotemplates. Specifically, we demonstrated a simple and robust method for the simultaneous patterning of differently labeled macromolecular structures onto a solid substrate. Hybridization-based programming of TMV provides an efficient means to greatly expand the addressing capability of nanotemplates under mild conditions and without the need for complex genetic or chemical modifications. Combined with the ability to functionalize these nanotemplates with metals, dyes, or antigens,1-5 this patterning method provides a powerful way to integrate functional macromolecular structures directly into nanoscale devices. We envision that a similar hybridization-based programming strategy could be further extended to the multiple assembly of other nanoscale entities. Specifically, one batch of nanomaterials could be manufactured with attached DNA of one sequence and then programmed to confer multiple functionalities and/or addresses via hybridization with linker DNAs in downstream processes.
Acknowledgment. We thank Tim Maugel in the Laboratory for Biological Ultrastructure for assistance with FE-SEM viewing. This work was supported in part by DOE Award DE-FG0202ER45975 and the Laboratory for Physical Sciences.
Figure 5. Scanning electron micrographs (SEM) of the differentially addressed TMV nanotemplates on DNA microarray platforms.
Figures 2-4 and confirm the specificity of the hybridizationbased programming and assembly. In summary, these results
Supporting Information Available: Fluorescence image of the entire microarray. This material is available free of charge via the Internet at http://pubs.acs.org. LA062493C
