NANO LETTERS
Nucleoprotein Assemblies for Cellular Biomarker Detection
2006 Vol. 6, No. 6 1184-1189
Elizabeth M. Singer* and Steven S. Smith City of Hope National Medical Center and Beckman Research Institute, 1500 East Duarte Road, Duarte, California 91010 Received March 10, 2006; Revised Manuscript Received April 17, 2006
ABSTRACT In this report, we have used DNA Y-junctions as fluorescent scaffolds for EcoRII methyltransferase-thioredoxin (M‚EcoRII-Trx) fusion proteins. Covalent links between the DNA scaffold and the methyltransferase were formed at preselected sites on the scaffold containing 5FdC. The resulting thioredoxin-targeted nanodevice was found to bind selectively to certain cell lines but not to others. The fusion protein was constructed so as to permit proteolytic cleavage of the thioredoxin peptide from the nanodevice. Proteolysis with thrombin or enterokinase effectively removed the thioredoxin peptide from the nanodevice and extinguished cell line specific binding measured by fluorescence. A number of potential applications for devices of this type can be envisioned. In particular, the ability of the fused protein to selectively target the nanodevice to certain tumor cell lines and not others suggests that this approach may serve as an adjunct to immunohistochemical methods in tumor classification as well as probe cell surface receptor architecture and function.
There has been a concerted effort in nanotechnology to develop bionanostructures as improved approaches to various biological applications. For example, advances in the field have begun to provide novel diagnostic tools that can help in disease screening and diagnosis. These approaches have included the use of biotinylated antibodies1 and peptides2,3 that have been linked to streptavidin and more complex structures such as quantum dots,4 fullerenes,5 and dendrimers.6,7 Detection has been accomplished by various means including fluorescence,8 paramagentism,9 and radioactivity.3 The biotin-avidin type of linking system has several disadvantages: the biotinylation conditions are harsh and not always effective, and there is little flexibility in the design. We are developing a novel linking system based on the DNA-methyltransferase-directed addressing of fusion proteins to DNA scaffolds.10-12 This linking system allows the display of multiple ligands at preselected positions on a DNA scaffold. The ligands can be either identical or nonidentical depending on the intended design. The DNA can be labeled with a variety of ligands, chromophores, or functional groups adding increased versatility to the system. The nucleic acid and protein components are all created separately and then assembled under physiological conditions. This allows the maximum degree of flexibility in the design and allows us to tailor the design of the nanodevice to the application. Of the available scaffolds, we have found the DNA Y-junction to be particularly useful. Unlike the four-way * To whom correspondence may be addressed. E-mail:
[email protected]. Phone: 626-359-8111, ext 64089. Fax: 626-301-8972. 10.1021/nl060549h CCC: $33.50 Published on Web 05/06/2006
© 2006 American Chemical Society
Figure 1. The three-dimensional structure of bacterial (left) and human (right) thioredoxin. Bacterial thioredoxin is structurally homologous to human thioredoxin, although it shares little sequence homology with its human counterpart. This figure was generated using InsightII (Accelrys, San Diego, CA) with the PDB coordinates of human (1AIU) and E. coli (1F6M) thioredoxin.
Holliday junction, even though the Y-junction has symmetry, it is stable because it is not capable of branch migration via Watson-Crick base pairing. Using this scaffold, we have generated a nanodevice which displays three copies of the bacterial thioredoxin (Trx) as cellular targeting ligand13 and carries one copy of the label fluoroscein on the DNA scaffold for detection. In this report we discuss molecular modeling used in the design of the thioredoxin targeted nanodevice, the analysis of its structure using microfluidics,10 its selective proteolytic cleavage, and its ability to differentially target living cells.
Figure 2. Model of the nanodevice. (A) DNA methyltransferase mechanism of action. During catalysis cytosine methyltransferases make a nucleophilic attack on C6 of cytosine or 5-fluorocytosine. This breaks the 5-6 double bond in the ring and activates C5 for methyl transfer. After the methyl group is transferred from S-adenosylmethionine (AdoMet) to C5, the normal progress of the reaction is to remove the hydrogen at C5 and the enzyme nucleophile from C6 by β-elimination. However, when fluorine is present at C5, this cannot occur because of the strength of the fluorine-carbon bond. Thus the progress of the reaction is halted and a covalent complex is formed between the enzyme and the cytosine ring targeted by the enzyme. In the case of M‚EcoRII, the second cytosine in the CCWGG recognition sequence is covalently bound to C(186) of the methyltransferase. (B) A homology model of M‚EcoRII was created as outlined below and detailed in the Supporting Information. This figure shows the position of the covalent bond between the active site cysteine (C186) and the C5 position in the methylated cytosine. The positions of hydrogen bonds between the cytosine and the amino acids phenylalanine 184 (F184), glutamic acid 233 (E233), and arginine 287 (R287) are also shown. This figure was produced using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco.27 (C) The model of the M‚EcoRII-Trx fusion protein coupled to double-stranded DNA. The M‚EcoRII-Trx fusion protein consists of the M‚EcoRII methyltransferase, a thioredoxin tag for targeting, a His6 tag for purification, and an S tag for detection. There is a thrombin cleavage site between the His6 tag and S tag and an enterokinase cleavage site between the S tag and the M‚EcoRII. We created a homology model of the C-terminal domain of M‚EcoRII with the PDB structure of M‚HhaI methyltransferase as a template using the Homology Module in Insight II (Accelrys, San Diego, CA). The N-terminal domain of M‚EcoRII has no known homology with any structure in the Protein Data Bank. We used GenTHREADER,28,29 a sequence-profile based fold recognition method available through the Protein Structure Prediction Server (PSIPRED)21,22 to predict the secondary structure. The linker region is composed of the His and S tags, and the sequences in between. We divided this region into segments (see Table 1) and searched for a similar structure for each segment individually. We used GenTHREADER to identify the structure of each segment in the nanodevice. The segments were connected by ligating the backbones and generating loops in the SWISSMODEL. The regions were manipulated by changing the Phi/Psi angles in residues at turns and in the connecting regions to prevent clashing. This linker region was then ligated onto the complete M‚EcoRII structure to form a model of the M‚EcoRII-Trx fusion protein substructure.
Several studies have implicated thioredoxin in the development of cancer. As a proof-of-concept demonstration of the potential utility of these devices in tumor classification, we have expressed bacterial thioredoxin, which is structurally homologous to human thioredoxin (Figure 1), although it shares little sequence homology with its human counterpart. In human cells thioredoxin is thought to be an inhibitor of protein-activated apoptosis.14 It is also exported,15 where it can serve as a cytokine.16 Thus, its continued expression and that of its associated interacting proteins (e.g., thioredoxin reductase) and transporters17 may be a hallmark of certain aggressive forms of prostate cancer. These properties suggest that the ability to detect this marker may be of significant value in tumor classification. The fundamental principle of the bionanotechnology used to assemble the nanodevices follows from the mechanism of action of the DNA (cytosine-5) methyltransferases.18,19 Like their cognate restriction enzymes, these enzymes recognize specific DNA sequences. In performing their natural function, they attack the cytosine ring in DNA and transfer a methyl group to the 5 position of the ring. Nano Lett., Vol. 6, No. 6, 2006
However, if they attack 5-fluorocytosine, they become covalently bound to the DNA molecule at their target sequence. (See Figure 2A.) Since 5-fluorocytosine can be introduced at any desired position in synthetic DNA, methyltransferases can be used to target fusion proteins to linear12,20 or branched DNA10 structures. The present nanodevice consists of three copies of the M‚ EcoRII-Trx fusion protein coupled to specific sites in the Y-junction DNA. The crystal structure of the M‚EcoRIITrx fusion protein, including the tags, has not been solved, so we generated a three-dimensional model of the nanodevice. We created a homology model of M‚EcoRII with the PDB structure of M‚HhaI as a template using the Homology Module in Insight II (see Supporting Information). A 5-fluoro-5-methyl 6-[C(186)-methyltransferase] cytosine intermediate was created in the model (Figure 2B). The N-terminal domain of M‚EcoRII has no known homology with any structure in the PDB database so we used Protein Structure Prediction Server (PSIPRED)21,22 to predict the secondary structure which was found to be mostly R helical. The M‚EcoRII sequence was threaded into the structure 1185
Figure 3. The EcoRII methyltransferase-thioredoxin fusion proteins were covalently coupled to the target sequences in the Y-junction DNA. The Y-junction DNA has three target sequences: one on each arm of the Y-junctions, so up to three EcoRII methyltransferasethioredoxin fusion proteins can be bound to one Y-junction. The Y-junction DNA is blue, the MecoRII-Trx fusion thioredoxin proteins are white, and the fluorescein is green. The molecular graphics images in this figure were produced using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco.27
using the SWISS-MODEL and the Swiss-PDB Viewer23 and then ligated onto the C-terminal domain of M‚EcoRII. The linker region is composed of the His and S tags, and the sequences in between. We divided this region into segments (see Table 1 in the Supporting Information) and searched for a similar structure for each segment individually. This linker region was then ligated onto the complete M‚EcoRII structure to form a model of the M‚EcoRII-Trx fusion protein substructure. Figure 2C shows the positions of all the segments of the M‚EcoRII-Trx fusion 1186
protein with the position of the tags and the cleavage sites. This model was placed onto the three 5-fluorocytosine-containing sites in the Y-junction DNA (Figure 3), yielding a computer-aided design for the structure that suggested that self-assembly of the nanodevice was possible. The M‚EcoRII-Trx fusion protein was cloned, expressed, and purified using the pET32a vector (Novagen, San Diego, CA) as described in the legend to Figure 4. The expressed protein has the following features: a thioredoxin tag for targeting, a His6 tag for purification, and an S tag for Nano Lett., Vol. 6, No. 6, 2006
Figure 4. Microfluidic separations of the Y-junction-coupled products. Y-junction-coupled products were detected using the newly developed microfluidics based electrophoretic mobility shift assay (EMSA) method available in the laboratory10 with the DNA 7500 LabChip. The results are depicted in virtual gel format. The three oligodeoxynucleotides have been shown to form a single Y-junction species in previous work.10 The unbound Y-junction has an apparent mobility corresponding to a 120 bp linear duplex (lane 1). The Y-junction containing three unoccupied M‚EcoRII sites, depicted graphically as a “Y”. Fluorescein was added to a central nucleotide in one of the arms of the Y-junction in a position, which did not interfere with M‚EcoRII binding. The fusion protein containing M‚EcoRII and thioredoxin (M‚EcoRII-Trx) was constructed using the same methyltransferase with individual inserts matched to the insertion sites by using PCR primers carrying restriction sites that orient the insert in frame with the M‚EcoRII. The E. coli expression vector pET32a was used. The plasmids containing the fusion proteins were transformed into BL21 competent cells and induced for 3 h with 1 mM IPTG when the cells reached a density of 1.5 OD600. The protein was expressed and purified from the cells as described in ref 10. M‚EcoRII-Trx is depicted as a sphere attached to the Y-junction with a fused Trx depicted as a smaller sphere attached to the M‚EcoRII by a linker region (line). Lane 2 is a virtual gel depiction of the microfluidicsseparated products of the nanodevice consisting of the Y-junction coupled to the M‚EcoRII-thioredoxin fusion protein. Addition of M‚Eco RII-Trx fusion produces mono-, di-, and trisubstituted Y-junctions with di- and trisubstituted forms dominating the products. The proteolytically cleaved nanodevice still generates three products, but each product is smaller than those of the uncleaved nanodevice. Lane 3 is a virtual gel depiction of the microfluidics separated products obtained following thrombin cleavage of the device. In this case, the Trx is removed leaving a linker depicted graphically by a line attached through a sphere (M‚ EcoRII) to the Y-junction. Lane 4 is a virtual gel depiction of the microfluidics-separated products obtained following enterokinase cleavage of the device; both the Trx and the linker are removed leaving a sphere (M‚EcoRII) attached to the Y-junction in the adjacent graphic.
detection. There is a thrombin cleavage site between the His6 tag and S tag, and an enterokinase cleavage site between the S tag and the M‚EcoRII (Figure 2C). This fusion protein was covalently bound to three specific sites in the DNA Y-junction and evaluated for coupling. The Y-junction DNAs were also generated as previously described10 except that the methyltransferase recognition sites present on each arm of the junction were the CFCWGG/CMNano Lett., Vol. 6, No. 6, 2006
CWGG sequence required by M‚EcoRII. 5-Fluorocytosine (FC) was introduced at the second cytosine on one of the strands and a methyl group (MC) was placed on the second cytosine in the complementary strand forming this site. The M‚EcoRII-Trx fusion protein was coupled to the Y-junction DNA as described.10 Figure 3 is a representation of a Y-junction with three M‚EcoRII fusion proteins bound. It also indicates the position of the Fluorescein (green) on one of the strands in the center of the junction, well away from the M‚EcoRII binding sites. We evaluated the assembly of the Y-junction coupled products by using a microfluidics based electrophoretic mobilty shift assay (EMSA) recently developed for this purpose in our laboratory10 (Figure 4). This allowed us to distinguish between free Y-junctions (Figure 4, lane 1) and Y-junction-coupled products with one, two, or three M‚ EcoRII fusion proteins bound (Figure 4, lane 2). We observed three products with the Y-junction, which has three M‚ EcoRII binding sites. The Y-junction coupled to M‚EcoRIITrx generates three products; with di- and trisubstituted forms dominating the products. The fusion protein was constructed so as to permit proteolytic cleavage of thioredoxin from the nanodevice as depicted in Figure 2C. Proteolysis with thrombin (Figure 4, lane 3) and enterokinase (Figure 4, lane 4) effectively removed the thioredoxin peptide from the nanodevice. Given the homology between the human and the bacterial thioredoxin, it was possible that the fluoroscene-containing nanodevice would bind to eukaryotic cells. We observed the binding properties of the fluorescently labeled Y-junction coupled products on MCF-7 cells grown on coverslips. The MCF-7 cells were exposed to solutions containing the nanodevice and evaluated by fluorescent microscopy. Fluorescent images of MCF-7 cells stained with the Y-junction coupled with M‚EcoRII-Trx obtained with a 400× objective are shown in Figure 5. The cells exposed to the Y-junction M‚EcoRII-Trx had a higher overall fluorescence and localized fluorescent signal around the cells. This indicates that the nanodevice may be binding to receptors in the surface membrane of the cells. We observed some background fluorescence of the cells exposed to phosphate-buffered saline (PBS) and Y-junction DNA alone (Y DNA) (Figure 5). When the thrombin or the enterokinase-treated nanodevice was exposed to MCF7 cells, fluorescence was extinguished (Figure 5) indicating that thioredoxin was the required targeting ligand. Given the properties of thioredoxin, it was possible that the fluoroscene-containing nanodevice would produce different signals with different cell lines. We tested the binding of the nanodevice on a variety of different cell lines including MCF-7, LnCaP, PC-3, COS-7, and primary prostate epithelial cells (PrEC). We observed that MCF-7 and LnCAP exposed to the Y-junction M‚EcoRII-Trx had a higher overall fluorescence and localized fluorescent signal around the cells. The PC-3, COS-7, and PrEC cells to the Y-junction 1187
Figure 5. Fluorescent images of cells exposed to the thioredoxin-targeted nanodevice. (A) MCF-7 cells were exposed to the nanodevice and assayed for cell surface binding using fluorescent microscopy. The cells were grown to 70% confluency directly on coverslips (Fischerbrand Coverglass for Growth Cover Glasses) and washed with ice cold PBS. After the wash, 300 nM of the fluorescently labeled ligand-targeted device and various controls were added to the cells in 400 µL of ice cold PBS. After 1 min of incubation, the supernatant was removed and cells were washed three times with ice-cold PBS. The coverslip was tapped dry to remove excess liquid. The cells were fixed by washing the coverlips with ice-cold methanol and placing them at -20 °C for 5 min. Each coverslip was mounted to a slide using Fluorescein Frag-EL mounting medium (Oncogene Research) and each coverslip was sealed with fingernail polish. The cells were observed and fluorescence was recorded at the Microscopy Core of the City of Hope Comprehensive Cancer Center by using an AX70 automated upright microscope using the U-MWIBA filter cube with a 460-490 excitation wavelength for fluorescence or Nomarski DIC (differential interference contrast). The images were taken with a cooled CCD color camera and processed with ImagePro Plus (Media Cybernetics, Inc., Silver Spring, MD) high-end image acquisition and analysis software. We observed localized fluorescence around the surface of the MCF-7 cells using fluorescent light with a FITC filter (panel 1). A differential interference contrast (DIC) image of the same cells is shown in panel 2. We observed some background fluorescence of the cells exposed to Y-junction DNA alone (panel 3) and to PBS (panel 4). We also observed decreased fluorescence of MCF7 cells exposed to the nanodevice after removal of the thioredoxin by proteolysis with thrombin (panel 5) and enterokinase (panel 6) indicating that thioredoxin was the required targeting ligand. (B) We observed differential binding of the fluorescent thioredoxin targeted nanodevice with different cell lines. Surface fluorescence was observed in LnCaP (panel 1) but not COS7 (panel 2), PC-3 (panel 3) or primary prostate epithelial cells (PrEC) (panel 4).
M‚EcoRII-Trx had a fluorescence similar to cells exposed to PBS (Figure 5). We have described an implementation of a general technology for the design and construction of nucleoprotein assemblies that has a broad spectrum of potential applications in molecular science. Many of these applications have been described previously.12,20,24-26 The nanodevice displaying three symmetrically clustered copies of thioredoxin described here, models a new class of assembly that can be envisioned for peptide and protein-ligand-based applications in cell and tumor biology as well as molecular science. For example, the expected increase in avidity afforded by multiple ligands suggests that the device could be useful in sorting cells based on surface marker expression type. The choice of ligand is limited only by the ability to express an appropriate fusion protein. Thus we are studying a second ligand-targeted device displaying copies of heregulin (unpublished data) for use in tumor cell classification. Moreover, the spatial orientation and the position of the 1188
ligand-fusion protein on the Y-junction DNA can be adjusted to determine the parameters associated with ligand display for optimal receptor interaction. The system can also be easily adapted to receptors for which ligand dimers are preferred since ligand dimerization can be achieved by close positioning on the scaffold or appropriate choices for the peptide linking the ligand to the methyltransferase. Although not explored in this study, methyltransferases targeting different recognition sequences12 can be employed on different arms of the Y-junction scaffold. Thus, ligandreceptor interactions could be studied by placing a ligand on one arm and a receptor domain on another. Such a system could be used to determine binding constants or to facilitate the development of inhibitors that disrupt receptor-ligand interaction. In addition, a device displaying two different ligands could be used as a probe for clustering of cell surface receptors. Our results provide a proof-of-concept demonstration that a nanoscale device comprising a DNA scaffold and three Nano Lett., Vol. 6, No. 6, 2006
thioredoxin fusion proteins can be constructed and used in cytological imaging. The results suggest that this particular device may be of value in prostate cancer diagnosis and staging since different prostate cancer cell lines image differently. Acknowledgment. Molecular graphics images were produced using the UCSF Chimera package from the Computer Graphics Laboratory, University of California, San Francisco (supported by NIH P41 RR-01081). This study was supported by the National Cancer Institute at the National Institutes of Health (NIH 5R01CA102521 and NIH 1F32CA110667). Supporting Information Available: Supplementary information provides a description of the homology modeling of M‚EcoRII, the construction of the linker, and the results of the minimization. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Kipriyanov, S. M.; Little, M.; Kropshofer, H.; Breitling, F.; Gotter, S.; Dubel, S. Protein Eng. 1996, 9 (2), 203-211. (2) Arttamangkul, S.; Alvarez-Maubecin, V.; Thomas, G.; Williams, J. T.; Grandy, D. K. Mol. Pharmacol. 2000, 58 (6), 1570-1580. (3) Boerman, O. C.; van Schaijk, F. G.; Oyen, W. J.; Corstens, F. H. J. Nucl. Med. 2003, 44 (3), 400-411. (4) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W.; Nie, S. Nat. Biotechnol. 2004, 22 (8), 969-9676. (5) Capaccio, M.; Gavalas, V. G.; Meier, M. S.; Anthony, J. E.; Bachas, L. G. Bioconjugate Chem. 2005, 16 (2), 241-244. (6) Shukla, S.; Wu, G.; Chatterjee, M.; Yang, W.; Sekido, M.; Diop, L. A.; Muller, R.; Sudimack, J. J.; Lee, R. J.; Barth, R. F.; Tjarks, W. Bioconjugate Chem. 2003, 14 (1), 158-167. (7) Choi, Y.; Thomas, T.; Kotlyar, A.; Islam, M. T.; Baker, J. R., Jr. Chem. Biol. 2005, 12 (1), 35-43. (8) Hsu, E. R.; Anslyn, E. V.; Dharmawardhane, S.; Alizadeh-Naderi, R.; Aaron, J. S.; Sokolov, K. V.; El-Naggar, A. K.; Gillenwater, A. M.; Richards-Kortum, R. R. Photochem. Photobiol. 2004, 79 (3), 272-279. (9) Mulder, W. J.; Koole, R.; Brandwijk, R. J.; Storm, G.; Chin, P. T.; Strijkers, G. J.; de Mello Donega, C.; Nicolay, K.; Griffioen, A. W. Nano Lett. 2006, 6 (1), 1-6.
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