EGFP Nanoparticle

Departments of Biochemistry, Microbiology and Immunology, Chemistry,. Oral Medicine, Chemical Engineering, and Clinical Pharmacy,. National Cheng Kung...
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NANO LETTERS

A Biological Strategy for Fabrication of Au/EGFP Nanoparticle Conjugates Retaining Bioactivity

2004 Vol. 4, No. 7 1209-1212

Chiau-Yuang Tsai,† Ai-Li Shiau,‡ Pai-Chiao Cheng,§ Dar-Bin Shieh,| Dong-Hwang Chen,⊥ Chen-His Chou,# Chen-Sheng Yeh,*,§ and Chao-Liang Wu*,† Departments of Biochemistry, Microbiology and Immunology, Chemistry, Oral Medicine, Chemical Engineering, and Clinical Pharmacy, National Cheng Kung UniVersity, Tainan 701, Taiwan Received March 28, 2004; Revised Manuscript Received May 2, 2004

ABSTRACT Recently, it has been found that thiolated oligonucleotides can be used to direct the assembly of gold colloids into ordered functional nanostructures. However, approaches for fabricating Au/DNA conjugates by using a linear, functional, double-stranded (ds) DNA fragment that directly binds to a Au NP surface have not been reported. In this paper, we used a ligase-dependent strategy to synthesize a 1.7 kb, thiolated dsDNA fragment and generated novel nanocrystals by coupling it to 13 nm Au NPs. The conjugates contained Au/EGFP with EGFP/ Au molar ratios of 1:1, 2:1, and 3:1, as determined by their electrophoretic mobility and AFM imaging. The Au/EGFP nanoconjugates could still be digested by restriction endonuclease (RE) and expressed as functional proteins in mammalian cells, indicating the biological activities of DNA fragments were retained after conjugation with Au NPs. This biological strategy for fabrication of Au/DNA conjugates may be applied to molecular imaging, nanomedicine, and nanobiosensor technology.

In recent years, it has been proposed that nanocrystal/DNA conjugates hold the promise for improving DNA array detection, biological imaging, and diagnostic technology.1-8 DNA/nanoparticle (NP) complexes have been synthesized by developing amine-modified NPs or utilizing electrostatic interaction to conjugate particle surfaces with various lengths of linear and plasmid DNA, and their biological activities have also been characterized.9,10 However, binding of Au NPs to a linear, functional, double-stranded (ds) DNA fragment has not been reported. Here, we demonstrate that dsDNA fragments containing a mammalian reporter gene expression cassette encoding the enhanced green fluorescent protein (EGFP) can be conjugated to 13-nm gold nanocrystals. Furthermore, the resulting nanoconjugates bearing discrete numbers of dsDNA fragments display normal functions of the DNA fragment, including site-specific digestion by restriction endonuclease (RE) and protein expression in mammalian cells. Despite recent reports regarding the conjugation of singlestranded (ss) DNA to Au NPs, few studies have documented * Corresponding author. E-mail: [email protected]; wumolbio@ mail.ncku.edu.tw Telephone: +886-6-235-3535; Fax: +886-6-274-1694. † Department of Biochemistry. ‡ Department of Microbiology and Immunology. § Department of Chemistry. | Department of Oral Medicine. ⊥ Department of Chemical Engineering. # Department of Clinical Pharmacy. 10.1021/nl049523l CCC: $27.50 Published on Web 05/25/2004

© 2004 American Chemical Society

the fabrication of Au/dsDNA conjugates.11 Holzel et al. obtained dsDNA carrying a 5′-thiol group by polymerase chain reaction (PCR) using 5′-thiol-labeled oligonucleotides as primers and linked it directly onto the gold electrodes.12 In this paper, we first developed a two-step, RE- and ligasedependent method for generating a 1714 base pair (bp), thiolated dsDNA fragment containing the EGFP expression cassette, designated S-EGFP, which was based on the joining of two individual fragments by T4 DNA ligase. The ligation reaction is ATP, Mg2+-dependent and its efficiency depends considerably on the length of overhangs used for annealing of oligonucleotides. The ligation-dependent technology is based on the construction of composite oligonucleotides from individual presynthesized blocks, such as representational difference analysis and ligation-dependent DNA cloning. Schematic presentation of the strategy for synthesizing alkylthiolated (S)-EGFP and fabricating Au/EGFP conjugates is illustrated in Figure 1. First, the 4.7 kb pEGFP-N1 plasmid (Clontech, Palo Alto, CA) was digested with AflII, dephosphorylated with calf intestine phosphatase, and then digested with AflIII. The resulting 1697 bp EGFP expression cassette encompassed the human cytomegalovirus immediate early promoter, EGFP cDNA, and SV40 early mRNA polyadenylation signal. A ds linker was produced by annealing two partially complementary single-stranded oligonucleotides, 5′-phophorylated (P)-CATGTTAATTAAGCT-

Figure 1. Schematic illustration of the strategy for the preparation of Au/EGFP conjugates.

TC, and 5′-alkylthiolated (S)-TTTTGAAGCTTAATTAA, which contained noncomplementary 4-nucleotide 5′-protruding single strands (overhangs). As the 5′-phosphorylated CATG overhang of the linker was complementary to the AflIII recognition site, it was ligated to the AflIII site of the EGFP fragment by ligase, resulting in the generation of S-EGFP with high efficiency (see Supporting Information). For preparation of Au/EGFP NP, S-EGFP was added into 13-nm Au NP solution and incubated for 24 h. The final

concentrations of DNA and colloid gold were 6.75 nM and 3.77 nM, respectively. Then, we used a 6 mer alkylthiolated oligonucleotide, designated protector (S6, 5′-S-ATCGAT), to fill void places on the Au NP surface. This process allowed the prepared Au/EGFP conjugates with high overall coverage of DNA oligomers, which enhanced their stability. As gel electrophoresis has been recently employed for isolation of colloidal gold nanocrystal/DNA conjugates,13,14 we applied Au/EGFP preparations to gel electrophoresis for detecting the fabrication of Au/EGFP NP. Figure 2a shows that Au/ EGFP conjugates were stable when subjected to agarose gel electrophoresis and migrated as two discrete bands of approximately 1.7 kb and 3.4 kb, indicating that 1 and 2 molecules of the EGFP DNA fragment were conjugated to 1 molecule of Au NP, which resulted in the fabrication of Au/1-EGFP and Au/2-EGFP, respectively. The Au/S6 conjugates serving as the negative control migrated much faster than Au/EGFP conjugates. Ethidium bromide and other intercalating dyes are widely used for fluorescent staining of DNA bands in agarose gels, which display a significant fluorescence enhancement (∼25-fold) upon binding to DNA. Discrete bands were visualized with gel electrophoresis using a 1% agarose gel and ethidium bromide via UV transmitters (Figure 2b). In addition, a thin band with higher molecular weight was observed, suggesting that one 13 nm Au NP was able to conjugate with more than two EGFP fragments. These results confirm that S-EGFP was successfully bound to Au NPs and discrete S-EGFP molecules onto Au NPs could be isolated by gel electrophoresis. Atomic force microscopy (AFM) imaging has been an important tool for studying DNA structures and DNAprotein interactions. Wang et al. observed the interaction between monolayer protected Au clusters (MPCs) and DNA chains by AFM.15 In this study we provide direct evidence

Figure 2. Electrophoretic mobility of Au/S-EGFP conjugates on 1% agarose gel. (A) Lane M corresponds to 100 bp DNA ladders. Lane 1 corresponds to 13-nm Au/S6 conjugates (single band). When S-EGFP fragments were added to the Au particles (lane 2), two discrete bands with estimated molecular weight of 1.7 kb and 3.4 kb (indicated by arrows) appeared, which contained Au/1-EGFP and Au/2-EGFP, respectively. The upper thin band, as indicated by arrow, may contain multiple molecules of the EGFP fragment conjugated to 1 molecule of Au NP. Lane 3 is the S-EGFP fragment serving as the unconjugated control. (B) Agarose gel stained with ethidium bromide. 1210

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Figure 3. The image of AFM surface plots of Au/EGFP conjugates. The red, blue, and green arrows indicate Au/1-EGFP, Au/ 2-EGFP, and Au/3-EGFP conjugates, respectively. The average diameter of the Au NP is 37.3 ( 2.8 nm.

to demonstrate that S-EGFP indeed bound to Au NPs as examined by AFM. Figure 3 illustrates the image of Au/ EGFP complexes where the bright dots represent Au NPs. Red, blue, and green arrows indicate Au/1-EGFP, Au/2EGFP, and Au/3-EGFP conjugates, respectively. The observed diameter of the Au NPs in the AFM image is larger than that measured in TEM, which is due to image convolution with the tip diameter.16 It has been shown that the biological activity of DNA interacting with Au colloids is partially retained.17 In fact, inhibition of its bioactivity has been observed in Au colloids.18 Nevertheless, Yun et al. showed that the bioactivity of DNA modified with 1.4-nm Au nanomaterials is unaltered.19 Here, we further investigated whether the bioactivity of DNA immobilized on 13-nm Au NPs could be maintained. Polyethylenimine (PEI) was introduced to join with Au/EGFP, resulting in the formation of Au/EGFP/PEI, a type of sandwich structure. The Au/EGFP/PEI complex was efficiently internalized into monkey kidney Cos-7 cells. Because our Au/EGFP complex was full of negative charges, addition of positive PEI was expected to neutralize the surface charge to achieve a net positive charge ratio and, hence, enhance transfection efficiency.20 EGFP expression was detected by fluorescent microscopy in the transfected Cos-7 cells (Figure 4), indicating that biologically active proteins could be expressed as encoded from linear dsDNA fragments containing mammalian expression cassettes, which were conjugated with Au colloids. To our knowledge, this is the first evidence to demonstrate that DNA fragments encoding proteins carried by Au NPs still retain their biological activity in mammalian cells. Finally, we show that digestion of Au/EGFP conjugates with RE resulted in the formation of smaller clusters, which ran faster than undigested conjugates on agarose gel electrophoresis (see Supporting Information), suggesting that the property of sitespecific digestion of the Au-conjugated DNA fragments by RE is unaltered. Nano Lett., Vol. 4, No. 7, 2004

Figure 4. Expression of the EGFP gene by Cos-7 cells transfected with DNA fragments encompassing EGFP expression cassette conjugated with gold colloids as monitored by (A) fluorescent and (B) bright field microscopy.

In summary, we describe a highly efficient strategy for producing gold nanocrystals linked with functional, dsDNA fragments and preparing discrete conjugates. Direct linkage between dsDNA and Au NPs was confirmed by both agarose gel electrophoresis and AFM measurements. The biological function of the conjugated DNA was maintained as determined by site-specific digestion by RE and expression of functional proteins in mammalian cells. Taken together, these results suggest that the fabrication of Au NPs directly linking with functional DNA fragments may allow for its applications on the design, preparation, and manipulation of broad architectures for molecular imaging, nanomedicine, or nanobiosensor technologies. Acknowledgment. This work was supported by grants from the National Science Council (NSC-92-2120-M-006002), Taiwan. Supporting Information Available: Analysis of S-EGFP production by agarose gel electrophoresis, cleavage of Au/ EGFP conjugates by RE, and some other photographs of 1211

fluorescence microscopy of Cos-7 cells transfected with Au/ EGFP conjugates are presented (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (2) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609611. (3) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071-9077. (4) Park, S.-J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 15031506. (5) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (6) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1540. (7) Glynou, K.; Ioannou, P. C.; Christopoulos, T. K.; Syriopoulou, V. Anal. Chem. 2003, 75, 4155-4160. (8) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 18841886. (9) Kneuer, C.; Sameti, M.; Bakowsky, U.; Schiestel, T.; Schirra, H.; Schmidt, H.; Lehr, C. M. Bioconjug. Chem. 2000, 11, 926-932.

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(10) McIntosh, C. M.; Esposito, E. A., III; Boal, A. K.; Simard, J. M.; Martin, C. T.; Rotello, V. M. J. Am. Chem. Soc. 2001, 123, 76267629. (11) Kanaras, A. G.; Wang, Z.; Bates, A. D.; Cosstick, R.; Brust, M. Angew. Chem. Int. Ed. 2003, 42, 191-194. (12) Holzel, R.; Gajovic-Eichelmann, N.; Bier, F. F. Biosens. Bioelectron. 2003, 18, 555-564. (13) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Alivisatos, A. P. Nano Lett. 2001, 1, 32-35. (14) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. J. Phys. Chem. B 2002, 106, 11758-11763. (15) Wang, G.; Zhang, J.; Murray, R. W. Anal. Chem. 2002, 74, 43207. (16) In our TEM data, the average size of Au NP is 13.1 ( 0.9 nm. (17) Gearheart, L. A.; Ploehn, H. J.; Murphy, C. J. J. Phys. Chem. B 2001, 105, 12609-12615. (18) McIntosh, C. M.; Esposito, E. A.; Boal, A. K.; Simard, J. M.; Martin, C. T.; Rotello, V. M. J. Am. Chem. Soc. 2001, 123, 7626-7629. (19) Yun, C. S.; Khitrov, G. A.; Vergona, D. E.; Reich, N. O.; Strouse, G. F. J. Am. Chem. Soc. 2002, 124, 7644-7645. (20) Ow Sullivan, M. M.; Green, J. J.; Przybycien, T. M. Gene Ther. 2003, 10, 1882-1890.

NL049523L

Nano Lett., Vol. 4, No. 7, 2004