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Chemical Modification of M13 Bacteriophage and Its Application in Cancer Cell Imaging Kai Li,†,‡ Yi Chen,‡ Siqi Li,‡ Huong Giang Nguyen,‡ Zhongwei Niu,‡ Shaojin You,§ Charlene M. Mello,| Xiaobing Lu,† and Qian Wang*,† State Key Laboratory of Fine Chemicals, Dalian University of Technology (DUT), Dalian, P. R. China, Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, Columbia, South Carolina 29208, Atlanta Research & Educational Foundation, Atlanta VA Medical Center 1670, Clairmont Road, Decatur, Georgia 30033, and Botulinum Research Center, University of Massachusetts, Dartmouth, 285 Old Westport Road, Dartmouth, Massachusetts 02747. Received September 14, 2009; Revised Manuscript Received April 12, 2010
The M13 bacteriophage has been demonstrated to be a robust scaffold for bionanomaterial development. In this paper, we report on the chemical modifications of three kinds of reactive groups, i.e., the amino groups of lysine residues or N-terminal, the carboxylic acid groups of aspartic acid or glutamic acid residues, and the phenol group of tyrosine residues, on M13 surface. The reactivity of each group was identified through conjugation with small fluorescent molecules. Furthermore, the regioselectivity of each reaction was investigated by HPLC-MSMS. By optimizing the reaction condition, hundreds of fluorescent moieties could be attached to create a highly fluorescent M13 bacteriophage. In addition, cancer cell targeting motifs such as folic acid could also be conjugated onto the M13 surface. Therefore, dual-modified M13 particles with folic acid and fluorescent molecules were synthesized via the selective modification of two kinds of reactive groups. Such dual-modified M13 particles showed very good binding affinity to human KB cancer cells, which demonstrated the potential applications of M13 bacteriophage in bioimaging and drug delivery.
INTRODUCTION In this paper, we report on our recent progress in fully screening the reactivity of three different reactive groups of the P8 coat protein of M13 phage. In addition, we also report on a potential application of the M13 phage in cell imaging to demonstrate how improved knowledge of its specific reactivity can be practically applied. M13 bacteriophage is a rod-like virus (880 nm long and 6.6 nm wide for wild-type) composed of a circular, single-stranded DNA that is encapsulated by approximately 2700 copies of the major coat protein P8 and capped with 5 copies of four different minor coat proteins (P9, P7, P6, and P3) on the ends (Figure 1) (1, 2). The functionality of these subunit proteins can be engineered to identify and display peptides that have a good binding affinity and specificity for essentially any target analyte through a technique called phage display (3). On the basis of this technology, a great number of M13 mutations were selected for many interesting applications, such as high-power phage batteries (2, 4, 5), tissue regenerating materials (6), metal nanowire catalysts (7), biological sensors and cell-targeting agents (8-11), gene transfer vectors (12, 13), and targeted cancer therapies (14). Furthermore, M13 bacteriophage has been used as a template to align inorganic, organic, and biological nanomaterials to generate different nanostructures, such as nanorings (15), nanowires (16, 17), films (18-22), nanofibers (1, 23), semiconductors (24), and metal hybrid materials (25). Although phage display is a powerful technology, * Correspondence to Qian Wang, Ph.D., Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208. Phone: ++001-803-777-8436. Fax: ++001-803-777-9521. E-mail:
[email protected]. † DUT. ‡ University of South Carolina. § Atlanta VA Medical Center. | University of Massachusetts.
it relies entirely on peptide expressions and selection. If the needed functionalities were beyond peptide modification, the genetic tools would fail to address the requirements. Orthogonal bioconjugation techniques have permeated fundamental virus chemistry, decorating addressable amino acids with a variety of molecules ranging from small molecules, such as fluorescent dyes, to large particles, such as quantum dots (26). As another simple way to generate functionalities, M13 bacteriophage has been chemically addressed with drugs (27-30), RGD peptides (31), near-infrared fluorescent dyes (32, 33), carboxylic acid groups (34), and other fluorophores (35, 36) for a variety of applications. Although these examples clearly showed that the P8 coat protein of M13 bacteriophage could be easily modified using simple bioconjugation methods, the detailed reactivity and regioselectivity of the reactive residues have not been reported yet. Therefore, it is necessary to fully investigate those two properties of all reactive groups on the M13 surface to further widen its potential application. Virus and virus-like particles (VLPs) have been currently used as scaffolds for bioimaging applications (37, 38). These bionanoparticles have the advantages of nanoscale size, high solubility, and the capability for multivalent and orthogonal display. Multivalency is important, since both the targeting and the signaling motifs could be displayed throughout the viral surface to significantly increase cellular uptake and signal intensity, respectively (39). Here, we showed that M13 phage could be modified simultaneously with multiple functional units, which could be used as a probe for cancer cell imaging.
EXPERIMENTAL SECTION General. Reactive fluorescein (FL) N-hydroxysuccinimidyl (NHS) ester and N,N,N′,N′-tetramethylrhodamine (TMR) NHS ester, Modified Lowry Protein Assay Kit, and Pierce Biotin Quantification Kit were purchased from Pierce and used without further purification. The other chemicals were purchased from
10.1021/bc900405q 2010 American Chemical Society Published on Web 05/25/2010
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Figure 1. (Left) Schematic depiction of M13 bacteriophage. (Middle) Surface crystal structure of M13 bacteriophage. (Right) The structure of M13 P8 protein subunit with target amino acid residues highlighted (59).
VWR and were used as received. Dialysis tubing of 300 000 molecular weight cutoff (MWCO) biotech was purchased from Spectrum Laboratories. TEM analyses were carried out by depositing 20 µL aliquots of each sample at a concentration of 0.1 mg/mL onto 100-mesh carbon-coated copper grids for 2 min. The grids were then stained with 20 µL of 2% uranyl acetate and viewed with a Hitachi H-8000 TEM electron microscope. Fluorescent microscopy was performed on Olympus IX 86 with 20× and 60× oil immersion objective lenses. MALDI-TOF MS of M13 P8 Subunit. A solution of M13 (1 mg/mL, 24 µL) was denatured by incubating with guanidinium chloride (6.0 M, 6 µL) for 5 min at RT, using Millipore ZipTipµ-C18 tips to remove the salts. The denatured protein was spotted onto a MALDI plate. These spots were analyzed by a Bruker Ultra-Flex I TOF/TOF mass spectrometer. MS-grade 2,5dihydroxybenzoic acid with 0.1% TFA was used as the matrix. HPLC-MS/MS Study. The mass spectra were acquired in the positive ion mode using a Micromass Q TOF spectrometer (Waters, MA) equipped with an electrospray source. The source temperature was held constant at 100 °C. The desolvation temperature was set at 350 °C. The capillary voltage was 3 kV. Full-scan spectra were collected from m/z 100 to 4000. The collision energy in the collision cell was set around 45 V. Masslynx 4.0 was used for data acquisition and analysis. Reactivity Screening of M13 Bacteriophage. The reactivity of the amino groups of M13 bacteriophage was screened by incubating with varying concentrations of TMR-NHS at a phage concentration of 1.0 mg/mL in a mixed solution of buffer and dimethyl sulfoxide (DMSO) (V/V ) 80:20). The reactions were performed at 4 °C for 24 h. Reactions were purified by four dialysis steps of 5 h each against 2 L of potassium phosphate buffer (pH 7.8, 10 mM). The carboxylic acid groups of M13 bacteriophage were screened for reactivity by incubating with varying concentrations of Rhodamine B amine (RB), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (10 mM), and N-hydroxysulfosuccinimide (Sulfo-NHS) (30 mM) in potassium phosphate buffer (pH 7.8, 10 mM) at room temperature for 12 h and purified by dialysis. The loading of dyes per phage was determined by UV-visible spectroscopy. Wild-type phage concentrations were determined by measuring the absorbance at 269 nm; virus at 0.1 mg/mL gives a standard absorbance of 0.38 (40). Modified phage concentrations were determined by Modified
Lowry Protein Assay Kit from Pierce. The average molecular weight of the M13 bacteriophage is 1.64 × 107 Da (34). Dye concentrations were obtained by measurement of absorbance at λmax (517 nm for TMR, 559 nm for RB), with molar absorptivity calibrated for each use by mixing known quantities of dye with M13 (1.0 mg/mL). The integrity of the modified M13 bacteriophage was determined by TEM. Reactive residues were identified by using HPLC-MS/MS. Reactions on Tyrosine Residues. The 3-ethynylbenzenediazonium (100 mM, 500 µL) was prepared by incubating p-toluenesulfonic acid (0.35 M, 400 µL) and 3-ethynylaniline (78 mg/mL, 75 µL), NaNO2 (207 mg/mL, 25 µL) on ice for 1 h. The diazo linkage was created by incubating M13 bacteriophage (20 mg/mL, 250 µL) with 20 µL of the 3-ethynylbenzenediazonium (0.1 M) in borate buffer (0.1 M, 4.73 mL, pH 9.0) for 2 h on ice. The reaction was purified by dialysis against Tris buffer (10 mM, pH 8.0). A following Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction can efficiently conjugate small molecules such as coumarin to the tyrosine residues. For a standard CuAAC reaction, azide (100 mM, 79 µL) and the solution of modified M13 bacteriophage (4 mg/mL, 250 µL) were mixed with CuSO4 (100 mM, 10 µL) and NaAsc (200 mM, 10 µL) in Tris buffer (10 mM, 651 µL, pH ) 8.0) and incubated at room temperature for 18 h. Next, phage precipitates were removed by centrifugation for 20 min at 13 500 rcf at 4 °C. The suspension was collected and purified by dialysis. The number of biotins per phage was obtained by the Pierce Biotin Quantification Kit. The integrity of the modified M13 bacteriophage was determined by TEM. Reactive residues were identified using HPLC-MS-MS. Dual Modification of M13 Bacteriophage. M13 bacteriophage (20 mg/mL, 200 µL) was first incubated with FLNHS (0.2 M, 20 µL) in a mixed solution of phosphate buffer (pH 7.8, 10 mM) and DMSO (V/V ) 80:20) to generate a 190 dye/phage nanoparticle (M13-FL). After dialysis, the dyemodified M13 was further modified with folate-azide (synthesis can be found in Supporting Information) by the tyrosine conjugation method mentioned above. TEM results showed that dual-modified M13 (FL-M13-FA) was still kept intact after the reaction. Cell Culture. HeLa contaminant cell line-KB cells were obtained from ATCC. They were grown continuously as a monolayer by using folate-free RPMI 1640 media with 10%
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Scheme 1. Modification of M13 Bacteriophage
FBS, penicillin (50 units/mL), streptomycin (50 µg/mL), and 2 mM L-glutamine at 37 °C in a 5% CO2/95% air humidified atmosphere. Cell Imaging Study. Cells were seeded into a 12-well plate containing 18 mm sterile glass coverslips at approximately 1 × 105 cells/well. After overnight incubation, the cells were exposed to FL-M13 (37 µL, 2.7 mg/mL), FL-M13-Folate (37 µL, 2.7 mg/mL), or FA-FL (37 µL, 2.7 mg/mL) (synthesis can be found in Supporting Information), respectively, with 963 µL of culture media from 1 to 24 h. Following incubation, cells were washed 3 times with folate-free media to remove unbound phage and then fixed with 4% paraformaldehyde in Dulbeccomodified phosphate buffer saline (DPBS) for 10 min. After fixing, the cells were washed 3 times with DPBS buffer and then stained with 4′,6-diamidino-2-phenylindole (DAPI) (5 µg/ mL, 1 mL) for 10 min. The cells were washed 3 times with DPBS and then mounted with GVA mount (VWR) on glass slides. The cells were examined with an Olympus IX 86 confocal microscope.
RESULTS AND DISCUSSION Reactivity of Amine Conjugation. M13 was purified in high yield from Escherichia coli cells (34). The integrity of virus through the purification was confirmed by TEM analysis (Supporting Information Figure S7A). TMR-NHS, which was selected for reactions with amino groups of protein, was utilized to study the reactivity of M13 bacteriophage (Scheme 1). Lysine reactive NHS chemistry was used. The reaction occurs via a
nucleophilic attack by an unprotonated amine on the ester, followed by an amide bond formation and release of NHS (41). For the protein labeling reaction, pH range 7.0-9.2 could be considered optimal conditions (42). Here, pH 7.8 buffer was selected for the reaction. In order to screen the reactivity of amino groups on M13 bacteriophage, different concentrations of TMR-NHS (40 µM to 8 mM) were used. The reactions were purified via dialysis and analyzed with UV-vis spectroscopy. The modified M13 bacteriophages were stable and remained intact as confirmed by TEM (Supporting Information Figure S7B). The normal absorption spectrum of free TMR is shown in Figure 2E (blue line). Compared to free dye, the modified bacteriophage (M13-TMR) displays two absorption peaks around 517 and 555 nm (red line). Whereas the 555 nm peak corresponds to the rhodamine monomers, the peak around 517 nm is caused by the formation of the tetramethylrhodamine dimers (43). The absorbance of free TMR at 280 nm gave noticeable differences in the spectra of wild-type M13 (black line) and M13-TMR complex (red line). The UV-vis measurements were used to calculate the dye per particle ratios based on the molar absorptivity of the dye and of the virus. When different concentrations of TMR-NHS were used, up to 1600 rhodamine units could be attached on to M13 bacteriophage, which gave a very high local concentration of fluorescent dye (Figure 2A). Since the fluorescent dyes were covalently linked to the virus particles at the defined position, we expected that the virus scaffold could prevent possible
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Figure 2. (A) Loading of TMR on M13 bacteriophage related to concentration of reagents in the reaction mixture. The loading of dye units per phage plateaus at a TMR-NHS ester concentration greater than 4 mM. Every data point displayed in this figure was based on multiple repeated experiments. The error bars in this figure represent the average values of standard deviations of all data points. (B) Overall fluorescence intensity vs dye loading for M13-TMR. The concentration of the phage in all samples in this study was controlled at 0.2 mg/mL. (C) The distance between the amino groups of alanine 1 and lysine 8 is 9.11 Å (Distance was measured by Pymol with a protein structure PDB file 1ifj). (D) MALDI-TOF MS of whole P8 subunit of M13 and M13-TMR. WT-M13: wild-type M13 coat protein, m/z 5,238. M13-TMR (single): monomodified M13 coat protein, m/z 5650. M13-TMR (double): double-modified M13 coat protein, m/z 6064 (the theoretical mass difference upon modification with TMR ) 412 Da). (E) UV-vis spectra showing bacteriophage absorbance of M13 (black curve), M13-TMR (red curve), and free dye TMR-NHS (blue curve) in solution. The viral concentrations were comparable for M13 and M13-TMR. The concentration of dye TMR-NHS was comparable for M13-TMR.
fluorescence quenching. Unfortunately, an obvious fluorescence quenching was detected. As shown in Figure 2B, the overall fluorescence intensity of M13-A began to decrease upon anchorage of more than ∼400 dye units per phage particle. Compared to similar amounts of the free dyes in solution, the fluorescence intensity of the M13-TMR complexes was lower (data not shown). The fluorescence intensity of M13-TMR was reduced because of the self-quenching that might have been caused by the close distance between rhodamine moieties, which could form nonfluorescent dimers (44). The proximity (9.11 Å) of two available modification cites (alanine 1 and lysine 8) could be a factor contributing to the formation nonfluorescent dimers (see the following discussion and Figure 2C) (45). The MALDI-TOF mass spectra showed the intense peak of protonated coat proteins of wild-type M13 at m/z 5238 (Figure 2D). At a lower level of modification (340 dyes per phage), single showed two major peaks, one at m/z 5238 from wildtype M13 and another at m/z 5650 resulting from the protonated monomodified coat protein of M13 with TMR (M13-TMR). The 412 m/z mass difference between wild-type M13 and M13-TMR was consistent with the theoretical mass of newly added TMR unit (Figure 2D). At a higher level of modification (600 dyes per phage), a third peak at m/z 6064 was observed, suggesting that some P8 proteins were doubly modified by TMR. As shown in Figure 1, one P8 subunit of M13 bacteriophage
contains five lysine groups (Lys 8, 40, 43, 44, 48) and one N-terminal amine on Ala 1. On the basis of the previous works reported on bioconjugation of bionanoparticles, it is usually regarded that amine modifications happen at exposed amine groups (28, 31, 34, 46-49). Thus, HPLC-MS/MS was employed to identify the modification sites. The MS data showed that at the lower levels of modifications only Ala-1 could be modified; however, at the higher levels of modification both Ala-1 and Lys-8 could be modified (detailed discussions can be found in the Supporting Information). Reactivity of Carboxylic Acid Residues. Four carboxylic acid groups are found on one P8 subunit: they are Glu 2 and 20 and Asp 4 and 5 (Figure 1). The reactivity of the carboxylic acid groups of M13 bacteriophage was screened by treating the viral particle with RB dye activated with EDC and sulfo-NHS (Scheme 1). Up to 150 carboxyl groups were derivatized after 12 h incubation (Figure 3B). As shown in Figure 3A, after conjugation, the modified bacteriophage (M13-RB) gave a dye absorbance at 559 nm. The MALDI-TOF mass spectra pointed to successful modification, represented by the peak of M13RB at m/z 5677 (Figure 3C). The modified M13 bacteriophages were stable and remained intact as confirmed by TEM (Supporting Information Figure S7C). Compared to amine conjugation, the carboxylic acid groups on M13 showed relatively low levels of activities toward RB, which made it harder to identify the reactive residues using
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Figure 3. (A) UV-vis spectra showing bacteriophage absorbance of wild-type M13 (black curve) and M13-RB (red curve) after purification. (B) Loading of RB on M13 bacteriophage as related to concentration of reagents in the reaction mixture. Every data point displayed in this figure was based on multiple repeated experiments. The error bars in this figure represent the average values of standard deviations of all data points. (C) MALDI-TOF MS of whole subunit of wild-type M13 (m/z 5238), M13-RB (m/z 5677), and M13-A (m/z 5277).
Figure 4. (A) MALDI-TOF MS of whole subunit of wild-type M13 and M13-B (m/z 5367), and a following conjugation with compound C (M13-C: m/z 5570) or compound D (M13-D: m/z 5693). (B) UV-vis spectra showing bacteriophage absorbance of wild-type M13 (black curve) and M13-C (purple curve). M13-C gives a strong absorbance at 340 nm.
HPLC-MS/MS. In order to solve this problem, prop-2-yn1-amine (compound A), a smaller molecule with better water solubility, was used in the reaction. The MALDI-TOF mass spectra of the coupling products can be found in Figure 3C. Similarly, HPLC-MS/MS was employed to uncover the specific reactive sites. Among all carboxylic acid groups, Glu-2 was reactive and Glu-20 was too “buried” to be modified. Asp-4/Asp-5 could also be modified, but it was difficult to determine which residue(s) contributed to the reactivity due to their proximity. Detailed analysis of the MS results can be found in Supporting Information. Tyrosine Conjugation. There are two tyrosine residues (Tyr 21 and Tyr 24) on the P8 protein (Figure 1). In order to test the possibility of tyrosine conjugation on M13 bacteriophage,
a diazonium coupling reaction with compound B was applied (Scheme 1) (50-52). As shown in Figure 4A, the modified peak can be found at m/z 5367, which indicates that the P8 protein was decorated with the alkyne moiety (M13-B). Although it was well-known that diazonium groups were able to react with a variety of protein functional groups, including tyrosine, histidine, or lysine residues (53-55), the HPLCMS/MS data indicated that the modifications only took place at tyrosine residues and both Tyr 21 and 24 were modified (See Supporting Information). A CuAAC reaction was then performed to conjugate azides to the alkynyl groups. As shown in Figure 4A, coumarin (compound C) and biotin (compound D) were successfully conjugated onto M13 surface based on MALDI MS analyses (M13-C at m/z 5570;
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Figure 5. (A) MALDI-TOF MS of whole subunit of WT-M13 and M13 after dual modification with FL-NHS and folic acid. Wild-type M13 (WT-M13): peak ) m/z 5238; the sequential modifications show the peaks of m/z 5596 and m/z 5890, respectively, indicating the fluorescein and then folic acid conjugation. (B) UV-vis spectra showing bacteriophage absorbance of wild-type M13 (black curve) and dual-modified M13 bacteriophage (orange curve) after purification. Dual-modified M13 bacteriophage shows an absorbance at 494 nm. (C) The surface structure of M13 bacteriophage with highlighting amine and tyrosine conjugations.
M13-D at m/z 5693). The biotin concentration was calculated using a Pierce Biotin Quantitation Kit, and around 400 biotins were conjugated per phage. The coumarin clicked compound can be easily monitored by UV-visible absorption at 340 nm (Figure 4B). Dual Modification on M13 Bacteriophage and Cell Imaging Study. Vitamin folic acid (FA) is one of the most common ligands for cancer cell targeting. It plays an important role in tissue growth and, at the cellular level, in cell division and DNA synthesis. Uptake of FA into cells is mediated by the folate receptor (FR), which is particularly overexpressed in many cancers compared to the limited distribution found on normal cells (56). On the basis of the preliminary conjugation analysis, the amino groups and tyrosine groups were chosen as reactive sites for the dual-modification M13 bacteriophage with fluorescent dyes and folic acid motifs. The two reactive groups were chosen because of their higher reactivities, and possible crosslinking caused by EDC chemistry could be avoided. Here, by combining amine and tyrosine bioconjugation, folic acid and fluorescent dye modified M13 bacteriophage could be readily prepared for application in cancer cell imaging. M13 was first treated with FL-NHS to get a 190-fluorescein-unit tailored bacteriophage. After purification, the dye-modified M13 could be further modified with azide-folate via a two-step tyrosine reaction. As shown in Figure 5A, the m/z 5596 peak indicated the fluorescent dye modification, and the peak at m/z 5890 indicated the dye and folate dual modified peak. On the basis
of the intensity of MS, it was estimated that around 400 folic acid molecules were successfully conjugated onto the M13 surface. The modified M13 bacteriophages were stable and remained intact as confirmed by TEM (Supporting Information Figure S7D). The dual-modified M13 bacteriophage was investigated for its potential application in tumor cell targeting. HeLa contaminant KB cells were used for the targeting study. Before the imaging study, the KB cells were cultured in the folate-free media to promote the overexpression of the folatebinding proteins. The cellular uptake of M13 bacteriophages in KB cells was analyzed using fluorescence microscopy. Besides dual-modified M13 bacteriophage (FA-M13-FL), two controls, M13-FL and small molecular probe FA-FL, were used. After 1, 8, and 24 h incubation of each sample with KB cells, the cells were fixed and images taken. As shown in Figure 6, the blue color represents cell nuclei, which were labeled with DAPI, and the green color came from different fluorescein-modified samples (M13-FL, FA-FL, and FA-M13FL). As expected, FA-M13-FL (Figure 6C) showed higher cellular uptake than the M13 bacteriophage that was only modified with fluorescein (M13-FL, Figure 6B). Furthermore, compared with FA-M13-FL particles, the small molecule probe, fluorescein-folate conjugate (FA-FL), only showed weak signals (Figure 6A). The cellular uptake of dualmodified M13 bacteriophage showed a significant increase after longer incubation time. Although the cytoplasm of the
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Figure 6. (A-C) Binding and internalization images of FA-FL, M13-FL, and FA-M13-FL into KB cells during different time periods. From left to right, images represent different fluorescent channels. Scale bar: 40 µm (confocal setting, 20×/0.85 oil lens; gain, 0; ND, 1; exposure time: cDAPI, 110 ms; cFITC, 500 ms). (D) Three-dimensional view of FA-M13-FL cell image. The top and left columns show the z-axis images. Scale bar: 10 µm (confocal setting, 60×/1.35 oil lens; gain, 0; ND, 1; exposure time: cDAPI, 30 ms; cFITC, 500 ms).
cells were not specifically stained, three-dimensional imaging indicated that the M13 bacteriophage might be located in the cytoplasm of KB cells (Figure 6D). Full z-stack 3D rendered rotational video images from confocal imaging are available in the Supporting Information.
CONCLUSION In conclusion, we fully screened the reactivity of three different reactive groups on the M13 surface by traditional bioconjugation methods. Amine-NHS coupling chemistry led to functionalization at N-terminus and Lys-8 residues, which was confirmed by LC-MS/MS, while the N-terminal amino group showed significantly higher reactivity than the amino group of the Lys-8 residue, which may be attributed to their different pKa (N-terminal R-amine: 7.6-8.0; ε-amine of lysine: 9.3-9.5) (57). As reported, the lower pKa value of N-terminal amine groups (relative to lysines) can be selectively modified in a lower pH reaction (58). Carboxylic acid groups and tyrosine residues of M13 could also be modified at a relatively lower level compared to amine group. For a tyrosine modification, a following “click” reaction could easily conjugate different small molecules onto the M13 surface. After the chemical modifications, the M13 particles were still kept intact, which was confirmed by TEM analysis. Through dual modification of M13 with fluorescent dyes and a cell targeting motif, such as folic acid, the newly functionalized biological particle could be used as fluorescent probe for human KB cancer cell imaging.
ACKNOWLEDGMENT We are grateful for the financial support from the US NSF (CAREER program and DMR-0706431), US DoD (W911NF09-1-0236), the Alfred P. Sloan Scholarship, the Camille Dreyfus Teacher Scholar Award, DoD-BCRP, and the W. M. Keck Foundation. We thank L. Andrew Lee and Elizabeth Balizan for the assistance of confocal images, and Laying Wu for the assistance of TEM images. Kai Li would like to thank the Scholarship from the China Scholarship Council. Huong Giang Nguyen would like to thank the Magellan Scholar Program from USC Research Foundation. We are also grateful for all constructive suggestions given by all reviewers. Supporting Information Available: MALDI spectra of wildtype M13 and chemically derivatized M13, and the MS/MS spectra of wild-type M13 with most of b-ions and y-ions marked, discussions of the site specificity and MS data, synthesis of all small molecules, and TEM images of M13 and its derivatives. This material is available free of charge via the Internet at http:// pubs.acs.org.
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