NANO LETTERS
Reversible and Chemically Programmable Micelle Assembly with DNA Block-Copolymer Amphiphiles
2004 Vol. 4, No. 6 1055-1058
Zhi Li, Yi Zhang, Paige Fullhart, and Chad A. Mirkin* Department of Chemistry and Institute for Nanotechnology, Northwestern UniVersity, 2145 Sheridan Road, EVanston, Illinois 60208-3113 Received March 7, 2004; Revised Manuscript Received April 6, 2004
ABSTRACT We report a general solid-phase synthesis strategy for a novel class of DNA block copolymer amphiphiles. The assembled spherical micelle structures exhibit recognition properties defined by their DNA sequences and can be used to build higher-ordered structures through hybridization with materials that possess complementary DNA. This work is an important step toward creating DNA modified structures that can act as phase transfer agents and carriers of nanovesicles.
Amphiphilic block copolymers that contain at least one hydrophobic polymer block and one hydrophilic block have been shown to generate ordered supramolecular structures such as monolayers, micelles, vesicles, bilayers, helices, and rod- and sheet-like structures in solution or at biphasic interfaces.1-8 Potential applications for these polymer amphiphiles include encapsulating agents for catalysis and drug delivery, surfactants for emulsions, adhesion promoters, and materials that can be used in chemical separation. Oligopeptides have been incorporated as blocks in such structures to provide scaffolding for assembly and subsequent chemical reactions within the larger supramolecular structures.8-10 Larger polypeptide amphiphiles with alternating polar and nonpolar regions have been explored for their structural properties,11,12 and recently micelle and vesicle structures have been synthesized from a novel type of “giant amphiphile” with a protein or enzyme as its hydrophilic group and a synthetic polymer as its hydrophobic group.13-14 Recently, significant research has focused on using DNA as a synthetically programmable interconnect for the preparation of new materials with preconceived architectural parameters and properties.15-17 Such materials have led to the development of new biological detection schemes,18 novel nanostructures,19 and the construction of nanoelectronic devices.20 Efforts also have been focused on the construction of DNA- polymer hybrid materials which have been explored for their potential in biodiagnostics and cellular uptake studies.21-24 Herein we report a strategy that bridges the prior work involving DNA-driven assembly of inorganic nanoparticles with the concepts used to generate organic micelle * Corresponding author. Fax: (+1) 847-467-5123. E-mail: camirkin@ chem.northwestern.edu. 10.1021/nl049628o CCC: $27.50 Published on Web 05/01/2004
© 2004 American Chemical Society
structures. Specifically, we report a general solid phase synthesis strategy and the subsequent assembly properties for a novel class of hybrid DNA-hydrophobic polymer amphiphiles. These assembled spherical micelle structures have recognition properties defined by the DNA sequence used to construct them and can be used to build higher-ordered structures through hybridization with materials that possess complementary DNA sequences. The amphiphiles were prepared through solid-phase synthesis on controlled pore glass beads (CPG) in a manner similar to conventional oligonucleotide synthesis. The key reagent required to prepare the targeted amphiphiles is a polystyrene phosphoramidite 1. Compound 1 was synthesized by reacting an alcohol-terminated polystyrene (Mn,avg ) 5.6 × 103, PDI ) 1.1)25 with chlorophosphoramidite in anhydrous CH2Cl2. The product can be precipitated from the reaction mixture by using anhydrous CH3CN. The 31P NMR spectrum of 1 shows two single resonances at 148.7 and 148.2, which are diagnostic of the expected diastereomers. Compound 1 was used to couple the polystyrene fragment to alcohol-terminated oligonucleotides directly off the CPG, Scheme 1. The coupling of 1 with the 5′ hydroxyl group of the oligonucletide strand (5′-A5-ATCCTTATCAATATT-3′) bound to the CPG is carried out using the “syringe synthesis technique”.26 After 3 h of coupling time, unreacted 1 is removed from the system by rinsing the CPG with 50 mL of CH2Cl2 and 50 mL of dimethylformamide (DMF). After ammonium hydroxide deprotection and cleavage steps, the desired polystyrene-DNA conjugate 2 is soluble and can be extracted from the CPG with DMF. For a typical 10 µmolscale solid-phase DNA synthesis, 300 to 600 OD (optical density at 269 nm in DMF, roughly 0.2 to 0.4 µmol) of the
Scheme 1.
A synthetic route to polystyrene-DNA amphiphilesa
a A pre-designed DNA strand on CPG is first synthesized using standard phophoramidite chemistry. Compound 1 is then coupled on the 5′ end of oligonucletide strand (5′-A5-ATCCTTATCAATATT-3′) bound to the CPG using the “syringe synthesis technique”. The desired polystyrene-DNA conjugate 2 can be extracted from the CPG with DMF after ammonium hydroxide deprotection and cleavage steps.
Figure 1. Gel electrophoretic migration-shift assay showing the migration bands for DNA-polymer conjugate 2 (lanes 2 and 3) and a DNA sequence (same as in conjugate 2) without polymer (lane 1) in 2% agrose gel.
Figure 2. Tapping mode AFM image showing the spherical micelle structures constructed from polymer-DNA amphiphiles. Typically, a drop of micelle solution (5 µL) was placed on an aminopropyltrimethoxysilane functionalized mica surface, sprayed with dry N2, wash with deionized water, and dried again with flowing N2.
final amphiphile product can be collected, and the molecular weight and structural assignment are confirmed by MALDITOF mass spectroscopy (Mn,avg ) 11.7 × 103, trans-3indoleacrylic acid as matrix, see Supporting Information). Further, the purity of the polystyrene-DNA conjugate has been assessed using a gel electrophoretic migration-shift assay. The conjugate 2 moves along the migration direction significantly slower than its DNA component, a consequence of the covalently attached polymer block as well as the existence of assembled structures (vide infra), Figure 1. Importantly, no DNA can be detected in the migration band of 2, consistent with the characterization of polystyrene and DNA as a chemically coupled entity. Due to its amphiphilic nature, the polystyrene-DNA conjugate 2 is able to form stable suspensions in various solvents including CH2Cl2, THF, DMF, and H2O. Note that DNA exhibits almost no solubility in CH2Cl2 and THF, and polystyrene is not soluble in water. To take advantage of the recognition properties of the oligonucleotide portion of
the molecule, we have focused our initial studies in water. In a typical micelle formation experiment, H2O (9 mL) was gradually added to a DMF solution of 2 (1 mL, 35 OD). The majority of the DMF was removed from the mixture by dialysis (MWCO ) 10K, 24 h). After dialysis, the solution was allowed to incubate at room temperature for 24 h. Centrifugation (5K rpm, 10 min) was used to remove heavily aggregated structures from the cloudy solution. This resulted in a clear solution containing the micelles formed from 2. The micelles have been imaged by tapping mode AFM, which reveals a dense layer of spherical particles with diameters primarily in the 13 to18 nm range, Figure 2. The size distribution of the DNA-polystyrene micelles has been measured in solution via dynamic light scattering, which is consistent with an average particle diameter of 16.4 nm (25% polydispersity, quadratic simulation). This suggests that the cores of the micelle assemblies are nearly solid, and almost all of the DMF was removed after dialysis. Similarly, a series of polystyrene-DNA amphiphiles, which vary in DNA
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Figure 3. (A) DNA directed sequence specific assembly of DNApolymer micelles and DNA modified gold nanoparticles. The assembled aggregates can be reversibly disassembled by heating them above the “melting temperature” of the duplex strand interconnects. (B) The sharp melting transitions (melting curves) as monitored by the surface plasmon band of the Au nanoparticles at 520 nm vs solution temperature. The red line shows the melting curve for the assemblies formed from the DNA micelles and gold nanoparticles modified with complementary DNA. The black line shows the melting curve for the assemblies formed from DNA micelles and gold nanoparticles modified with single base mismatch DNA strands.
sequence length (5 mer, 10 mer, and 25 mer) and polystyrene molecular weight (4.1K, 7.2K, and 9.5K) yield micelle structures with tailorable average diameters in the 8 to 30 nm range. Note that a small amount of cylindrical rod structures occasionally can be found within the samples of spherical micelles (see Supporting Information). These micelles exhibit unique sequence-specific recognition properties, which derive from their hydrophilic DNA shells. Therefore, one can utilize them for the rational assembly of nanostructures through DNA hybridization. DNA-modified gold nanoparticles have been widely used as inorganic building blocks for the construction of many new types of superstructures.16,17 These micelle structures also can be used as novel organic building blocks in analogous schemes, allowing us to bridge the properties of the inorganic compositions with those of the micelle structures. For example, upon mixing a solution of 2 with a solution of 13 nm Au nanoparticles modified with complementary DNA (3′-TAGGAATAGTTATAA-A5-SH-5′) in 0.3 M NaCl, 10 mM phosphate buffer solution (pH ) 7.0), sequence specific assembly takes place. Consistent with DNA guiding this process, the aggregates can be reversibly disassembled by heating them above the “melting temperature” (Tm ) 57.8 °C) of the duplex strand interconnects. This results in a sharp melting transition as monitored by the surface plasmon band Nano Lett., Vol. 4, No. 6, 2004
of the Au nanoparticles at 520 nm, Figure 3. Note the disassembly process for this type of structure is highly sequence dependent. In a separate experiment, the “melting temperature” of aggregates formed from 2 and 13 nm Au nanoparticles modified with DNA strands that possess a single base mismatch (3′-TAGGAATATTTATAA-A5-SH5′) is 2.6 degrees lower (55.2 °C) than for the aggregates formed from the perfectly complementary strands. The origin of these sharp melting transitions (fwhm ) 2-3 °C) has been explained by a cooperative melting model described elsewhere for analogous gold nanoparticle and comb polymer systems.23,27 This work is important for the following reasons. (1) It provides a general method for preparing a new and versatile type of polymer-DNA amphiphile through solid-phase DNA synthesis. (2) The organic micelle structures generated from polymer-DNA amphiphiles have recognition properties defined by the DNA sequence used to construct them and can be used to build higher-ordered structures through sequence-specific hybridization with nanomaterials that possess complementary DNA strands. Finally, (3) this is an important step toward creating DNA modified structures that can act as phase transfer agents and carriers of nanovesicles. Acknowledgment. C.A.M. acknowledges the AFOSR (F 49620-02-0180) and NSF MRSEC of Northwestern University (DMR-0076097) for research support. The authors also acknowledge the use of the Dynamic Light Scattering instrument in the Keck Biophysics Facility at Northwestern University. Supporting Information Available: MALDI-TOF spectrum of 2 and image of cylindrical rod structures occasionally found within the samples of spherical micelles This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Discher, D. E.; Eisenberg, A. Science 2002, 297, 967-973. (2) Discher, B. M.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Curr. Opin. Colloid Interface Sci. 2000, 5, 125-131. (3) Cornelissen, J. J. L. M.; Donners, J. J. J. M.; de Gelder, R.; Graswinckel, W. S.; Metselaar, G. A.; Rowan, A. E.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science 2001, 293, 676-680. (4) Amphiphilic Block Copolymers: Self-assembly and Applications; Alexandridis, P., Lindman, B., Eds.; Elsevier Science: Amsterdam, 2000. (5) Cornelissen, J. J. L. M.; Fischer, M.; Sommerdijk, N. A. J. M.; Nolte, R. J. M. Science 1998, 280, 1427-1430. (6) Shen, H. W.; Eisenberg, A. Angew. Chem., Int. Ed. 2000, 39, 33103312. (7) Zhang, L. F.; Yu, K.; Eisenberg, A. Science 1996, 272, 1777-1779. (8) Stupp, S. I.; LeBonheur, V.; Walker, K.; Li, L. S.; Huggins, K. E.; Keser, M.; Amstutz, A. Science 1997, 276, 384-389. (9) Hartgerink, J. D.; Beniash E.; Stupp S. I. Science 2001, 194, 16841687. (10) Vauthey, S.; Santoso, S.; Gong, H.; Zhang, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5355-5360. (11) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell, D. A. Science 1998, 281, 389-392. (12) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J. Nature 2002, 417, 424-428. (13) Velonia, K.; Rowan, A. E.; Nolte, R. J. M. J. Am. Chem. Soc 2002, 124, 4224-4225. 1057
(14) Boerakker, M. J.; Hannink, J. M.; Bomans, P. H. H.; Frederik, P. M.; Nolte, R. J. M.; Meijer, E. M.; Sommerdijk, N. A. J. M. Angew. Chem., Int. Ed. 2002, 41, 4239-4241. (15) Storhoff, J. J.; Mirkin, C. A. Chem. ReV. 1999, 99, 1849-1862. (16) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (17) Alivisatos, A. P.; Johnsson, K. P.; Peng, X. G.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Nature 1996, 382, 609611. (18) Cao, Y. C.; Jin, R.; Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 14676-14677. (19) Seeman, N. C. Science 2003, 421, 427-431 (20) Keren, K.; Berman, R. S.; Buchstab, E.; Sivan, U.; Braun, E. Science 2003, 302, 1380-1382. (21) KorriYoussoufi, H.; Garnier, F.; Srivastava, P.; Godillot, P.; Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388-7389.
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(22) Thompson, L. A.; Kowalik, J.; Josowicz, M.; Janata, J. J. Am. Chem. Soc. 2003, 125, 324-325. (23) Watson, K. J.; Park, S. J.; Im, J. H.; Nguyen, S. T.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 5592-5593. (24) Jeong, J. H.; Park, T. G. Bioconjugate Chem. 2001, 12, 917-923. (25) van Hest, J. C. M.; Delnoye, D. A. P.; Baars, M. W. P. L.; ElissenRoman, C.; van Genderen, M. H. P.; Meijer, E. W. Chem.Eur. J. 1996, 2, 1616-1626. (26) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (27) Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643-1654.
NL049628O
Nano Lett., Vol. 4, No. 6, 2004
