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Molecular Fabrication: Aligning DNA Molecules as Building Blocks Masatoshi Ichikawa,† Yukiko Matsuzawa,‡ Yoshiyuki Koyama,§ and Kenichi Yoshikawa*,† Department of Physics, Graduate School of Science, Kyoto University and CREST, Kyoto 606-8502, Japan, and Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan, and Department of Home Economics, Otsuma Women’s University, 12 Sanban-cho, Chiyoda-ku, Tokyo 102-8357, Japan Received February 26, 2003. In Final Form: April 14, 2003 We report here a novel method for assembling macromolecules in a sequential order. Individual DNA molecules can be prefabricated into a linear micrometer-sized pearling chain, or colored bar, under trapping with a focused IR laser, by using a suitable chemical agent, Chol-PEG-A (poly(ethylene glycol) with aminoand cholesteryl-pendant groups), to complex with DNA. Using the laser, these fabricated bars can then be pieced together into a complex structure. This approach could be useful for the further development of micromanufacture using macromolecules as nanoelements.
Introduction There has been increasing interest in the fabrication of molecules into various complex patterns through the use of “nanotechnology”.1-6 For example, the transfer of ink containing chemically cogitated oligonucleotide to a solid plate has been used to draw linear arrays of DNA aggregates.6 Furthermore, recent technical developments have enabled the manipulation of single macromolecules,7-12 for example, the force-extension measurement of DNA and proteins by using optical trapping13-15 of a microbead attached to the end of the macromolecule. Although this optical technique has often been used in recent biophysical studies, chemical modification of the chain end is almost unavoidable.11,12 Quite recently, it has been reported that individual giant DNA molecules can be selectively trapped by a focused IR laser without any chemical modifications,16-20 when the molecular * To whom correspondence should be addressed. E-mail:
[email protected]. ac.jp. † Kyoto University and CREST. ‡ Toyohashi University of Technology. § Otsuma Women’s University. (1) Sleytr, U. B.; et al. FEMS Microbiol. Rev. 1997, 20, 151. (2) Seeman, N. C. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 225. (3) Prokop, A. Ann. N. Y. Acad. Sci. 2001, 944, 472. (4) Zasadzinski, J. A.; Kisak, E.; Evans, C. Curr. Opin. Colloid Interface Sci. 2001, 6, 85. (5) Keren, K.; et al. Science 2002, 297, 72. (6) Demers, L. M.; et al. Science 2002, 296, 1836. (7) Scha¨fer, B.; et al. Cytometry 1999, 36, 209. (8) Bustamante, C.; Macosko, J. C.; Wuite, G. J. L. Nature Rev. Mol. Cell. Biol. 2000, 1, 130. (9) Ishijima, A.; Yanagida, T. Trends Biochem. Sci. 2001, 26, 438. (10) Zlatanova, J.; Lindsay, S. M.; Leuba, S. H. Prog. Biophys. Mol. Biol. 2000, 74, 37. (11) Allemand, J. F.; Bensimon, D.; Jullien, L.; Bensimon, A.; Croquette, V. Biophys. J. 1997, 73, 2064. (12) Shivashankar, G. V.; Feingold, M.; Krichevsky, O.; Libchaber, A. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7916. (13) Gordon, J. P. Phys. Rev. A 1973, 8, 14. (14) Ashkin, A.; Dziedzic, J. M.; Bjorkholm, J. E.; Chu, S. Opt. Lett. 1986, 11, 288. (15) Svoboda, K.; Block, S. M. Annu. Rev. Biophys. Biomol. Struct. 1994, 23, 247. (16) Chiu, D. T.; Zare, R. N. J. Am. Chem. Soc. 1996, 118, 6512. (17) Matsuzawa, Y.; et al. J. Am. Chem. Soc. 1999, 121, 11581. (18) Yoshikawa, Y.; Nomura, S.-i.; Kanbe, T.; Yoshikawa, K. Chem. Phys. Lett. 2000, 330, 77.
chains are folded into a compact state or situated in a suspension of microbeads. It has been found that the folding transition is reversible; that is, collapsed DNA molecules unfold when they are optically transported to a different chemical medium.18 In the present study, we found that individual intact DNA molecules folded by a cationic synthetic polymer21-23 can be assembled into a micrometer-scale ordered structure by using single-beam laser trapping without any serious treatment, such as covalent bond formation. Materials and Methods Preparation of Folded DNA. T4 phage genome dsDNA chains (166 kbp, ∼56 µm, Nippon Gene) stained with DAPI (4′,6diamidino-2-phenylindole, Wako Pure Chemical Industries, Ltd) or YOYO-1 (1,1′-4,4,7,7,-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene]quinolinium tetraiodide, Molecular Probes) were used. Individual DNA molecules were folded by poly(ethylene glycol) derivatives with either amino-pendant groups (PEG-A) or both cholesteryland amino-pendant groups (Chol-PEG-A).21,23 The final concentrations, dissolved in MilliQ water (Millipore), were as follows: 0.60 µM DNA in base pairs, 0.60 µM DAPI or 0.10 µM YOYO-1, and 6.0 µM PEG-A or Chol-PEG-A (amino group concentration). Due to the blurring effect on the order of 0.3 µm, the apparent size in the fluorescent image is greater than the actual size. By directly measuring the Brownian motion of individual DNA molecules, the hydrodynamic radii of the particles were evaluated to be between 50 and 200 nm. These sub-micrometer-sized particles are stable enough to avoid spontaneous aggregation, reflecting a charged colloidal nature. Microscopy and Optical Trapping. Experimental observations and optical trapping were performed using a fluorescent microscope (Nikon) with a large-aperture oil-immersion objective lens (Plan Fluor ×100, NA ) 1.30, Nikon). A Nd:YAG laser (TEM00, CW 1064 nm, Spectron) for optical trapping was (19) Hirano, K.; Baba, Y.; Matsuzawa, Y.; Mizuno, A. Appl. Phys. Lett. 2002, 80, 515. (20) Matsuzawa, Y.; Hirano, K.; Mizuno, A.; Ichikawa, M.; Yoshikawa, K. Appl. Phys. Lett. 2002, 81, 3494. (21) Koyama, Y.; et al. Bioconjugate Chem. 1996, 7, 298. (22) Yoshikawa, K.; Yoshikawa, Y.; Koyama, Y.; Kanbe, T. J. Am. Chem. Soc. 1997, 119, 6473. (23) Koyama, Y.; Ito, T.; Kimura, T.; Murakami, A.; Yamaoka, T. J. Controlled Release 2001, 77, 357.
10.1021/la034338t CCC: $25.00 © 2003 American Chemical Society Published on Web 05/29/2003
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Figure 1. Formation of a molecular rod by alignment of folded compact DNAs using an infrared laser. (A) Elongated coils of T4 dsDNA are folded into compact globules by the addition of PEG-A (shown in photo) or Chol-PEG-A. These compact DNAs behave as soluble colloidal particles. (B) Electron micrographs of compact DNA molecules. (left) The complex with PEG-A has a quasispherical shape. (right) The complex with Chol-PEG-A shows characteristic small granules on the surface of a quasi-spherical structure. These granules are attributable to oily droplets composed of the cholesteryl moieties of Chol-PEG-A. (C) The assembly procedure, together with the fluorescent microscopic images. Top (laser on): Single DNA globules folded by PEG-A and Chol-PEG-A are optically trapped at the beam focus. Middle (collection): By moving the focus in the DNA solution, 9 and 17 compact DNA globules are collected, respectively. Bottom (laser off): When the laser is turned off, the assembly of DNA globules with PEG-A dissolves into individual globules, while the assembly of DNA complexed with Chol-PEG-A retains its shape. The DNA rod can be optically transported onto a glass surface, as shown in the bottom right picture. introduced into the objective lens by a dichroic mirror and focused to a point of about 1 µm on the observation field oriented to give a convergent angle of about 90-120°. We used an output laser power of between 500 and 1000 mW. An acousto-optic modulator (BRIMROSE) and micromotion control stage (SIGMA KOKI) were used for the optical and mechanical manipulation15 in Figure 3. Microscopic fluorescent images were detected by a highsensitivity SIT video camera and recorded through an image processor (HAMAMATSU Photonics). Electron microscopy experiments were carried out using negative staining with 1% uranyl acetate on a transmission electron microscope (JEOL) at 100 kV. The experiments were carried out at a room temperature of about 20 °C. Method of Simulation. Laser force can be described as a combination of an optical gradient force and a pushing force. When an object is near the focus of the laser trap, it is attracted to the focus, due to the trapping potential, along the direction of the light pressure. The stability and steric structure of the assembly are determined by three factors: (i) pair interaction between the particles, (ii) the shape of the trapping potential (convergence angle), and (iii) the strength of the pushing force. In the initial stage, a particle (seed of an aggregate) is located at the focus (r ) (0,0,0)). The motion of a guest particle is given by the overdamped Langevin equation:
γ
dri ∂U ) Ri(t) dt ∂ri
(1)
where γ is a friction coefficient of the particle. Ri(t) is Gaussian white noise and obeys the fluctuation-dissipation theorem. U is the total potential as the sum of the gradient force of light and
pressure.15 The average strength of the electric field 〈E(r)〉 of the laser light at r is assumed to be
(
)
-2(x2 + y2) 2 exp 2 〈E(r)2〉 ) ZP 2 -12 π(z + 0.25 × 10 ) z + 0.25 × 10-12
(2)
where the constants Z and P are the impedance and beam power, respectively. The radius of the beam waist (0.5 µm) corresponds to the diffraction limit of the 1064 nm laser. The aggregate is assumed to be a rigid body with regard to its translational and rotational motion. Time steps are regulated from 10-6 to 10-7 s depending on the trapping force. The time steps for particles far from the assembly are set at 0.001 to accelerate the calculation. In the above conditions, particles are pulled toward the focus one by one. The optical trapping force used in the simulation was consistent with the experimental value.17
Results and Discussion We prepared two different kinds of colloidal particles of T4DNA (166 kbp), compacted using PEG-A (Figure 1A) and Chol-PEG-A, respectively, with radii of 50-200 nm in aqueous solution as evaluated from electron micrographs (Figure 1B) and from the measurement of Brownian motion. The DNA concentration was 0.6 µM in base pairs.24-26 Figure 1C shows the procedure used to assemble (24) Sikorav, J. L.; Pelta, J.; Livolant, F. Biophys. J. 1994, 67, 1387. (25) Bloomfield, V. A. Biopolymers 1997, 44, 269.
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Figure 2. Simulation of the assembly of DNA globules with Brownian dynamics.31 For simplicity, we consider spherical particles with a radius of 100 nm. The particles are repulsive (upper) and adhesive (lower), as shown in the profile of the potential. The optical trapping laser is irradiated from -z to the origin with a convergence angle of 90°. The scale and the interaction between globules are depicted in the lower right panel. (A) Individual particles, exhibiting a colloidal nature, assemble under continuous laser irradiation. After the laser is turned off, the densely packed aggregate disintegrates into individual globules. The number of particles in each picture is 4, 11, 17, 17, and 18. (B) Sticky particles form an assembly with a linear arrangement. Since the particles always approach the focus from -z due to the optical pushing force, the trapped assembly develops toward +z. The assemblies include 4, 8, 12, 13, and 20 particles, respectively. (C) The geometry of the experimental setup. The depicted optical cones indicate the region with an intensity above e-2 in Gaussian laser light.
DNA molecules in prefabrication. The focused laser, with a power of about 500 mW, can achieve steady trapping of a desired folded compact DNA molecule.16,17 For the PEG-A complex, a quasi-spherical aggregate is formed through the collection of multiple DNA molecules, similar to the optical aggregation of a synthetic polymer.27 When the laser light is turned off, the assembly dissociates into individual DNA particles (bottom left panel in Figure 1C), reflecting the natural repulsion between the particles. In contrast, the DNA globules prepared with Chol-PEG-A are integrated into a rod-shaped assembly that grows along the optical cone. The ordered assembly can be transported to the glass substrate, as shown in the final panel in Figure 1C. The instability of the DNA/PEG-A assembly indicates that the globules do not stick to each other. On the other hand, DNA molecules compacted by Chol-PEG-A bind to each other; that is, they attract each other when in contact but repel each other when separated by a long distance because of their colloidal nature.21-23 We have confirmed that individual DNA particles folded by PEG-A and CholPEG-A are almost neutral and may be surrounded by a small amount of negative charge on the surface (data not shown).28 The sticky interaction of the Chol-PEG-A complex can be attributed to the hydrophobic interaction of small “oil droplets” made of cholesteryl moieties, as shown in the electron micrograph (Figure 1B right). The large difference in the stability of the assemblies (Figure 1C) can be explained in terms of the potential profile. It has been well established that compact globules made from single giant DNA molecules behave as charged colloidal particles; that is, the compaction of a single DNA
is a quite different phenomenon from the condensation/ precipitation of DNA molecules.29 Since the use of laser trapping or optical tweezers attracts dielectric objects due to the gradient of light intensity and exerts pressure along the optical path due to the momentum of photons, individual DNA particles tend to point along the direction of the incident laser beam. It is also known that a rodshaped object with a length greater than the wavelength of light is aligned along the optical axis.30 Thus, through the accumulation of individual DNA globules complexed with Chol-PEG-A, a rod of 10 µm or more in length is formed from many DNA molecules in a spontaneous manner. This rod is rather soft and can be bent by optical manipulation, as shown in Figure 1C. To obtain further insight into the differences between the physicochemical properties of the assemblies with PEG-A and Chol-PEG-A, we performed a simulation. Figure 2A and B shows the assembly of particles under a converged Gaussian laser beam, where the particles are repulsive and sticky at the contact length, respectively. For simplicity, we used the potential profile shown in the bottom right graph in Figure 2. When the particles are repulsive at contact, they form a densely packed aggregate, as in the crystal phase of a colloid. With the laser turned off, the particles repel each other and dissolve into the bulk phase, which corresponds to the experimental results with the DNA/PEG-A complex (Figure 1C left). In contrast, when the particles attract each other at the contact length, the assembly grows along the optical axis to form a long rodlike structure. Even after the laser is turned off, the rod-shaped assembly remains stable in solution, corre-
(26) He, S.; Arscott, P. G.; Bloomfield, V. A. Biopolymers 2000, 53, 329. (27) Hofkens, J.; Hotta, J.; Sasaki, K.; Masuhara, H.; Iwai, K. Langmuir 1997, 13, 414. (28) The effective surface charges of the DNA/PEG-A complexed particles were evaluated by the direct microscopic observation of electrophoresis in agarose gel (0.2%).
(29) Yoshikawa, K.; Yoshikawa, Y. In Pharmaceutical Perspectives of Nucleic Acid-Based Therapeutics; Kim, S. W., Mahato, R. I., Eds.; Taylor & Francis: London, U.K., 2002; Chapter 8. (30) Nomura, S.-i. M.; Harada, T.; Yoshikawa, K. Phys. Rev. Lett. 2002, 88, 093903. (31) Allan, M. P.; Tildesley, D. J. Computer Simulation of Liquids; Clarendon: Oxford, U.K., 1987.
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that the essential conditions for a straight rod are particle interaction that is sticky with dispersibility, and a gradient force with a shape and intensity that are adequate for controlling the collision of particles. Additionally, the pushing force exerted by the optical pressure plays a role in growth at the bottom of the object during assembly. Figure 3 shows the formation of a sequential arrangement and the fabrication of a molecular emblem using a complex of DNA with Chol-PEG-A. The fluorescent pictures in Figure 3A, together with the corresponding schematic drawings, show long rods composed of different colored blocks, where the blue and green regions correspond to assembles of DNA molecules stained with DAPI and YOYO-1, respectively. The procedure used to obtain these composites is illustrated at the bottom of Figure 3A; the laser focal point is moved between the two different regions stained with DAPI and YOYO-1. After composite structures are formed, the rod is optically transported to a glass surface, as shown in the pictures. To demonstrate the transport and orientation of DNA rods, we formed a pattern on the plate using the DNA rods. Figure 3B shows the character for “Kyo”, which is commonly used to refer to the city of Kyoto, constructed using the present experimental technique. During construction, nine DNA rods are connected to fabricate the symbol, where individual rods are prepared along the optical cone in the same solution. Figure 3. Construction of structures with DNA globules. (A) DNA globules stained with different dyes are assembled to form rods under laser trapping (see Figures 1 and 2). After the desired DNA globules are collected, the rod-shaped object is transported onto a glass surface. Blue and green colors are fluorescence from DAPI and YOYO-1, respectively. The blue and green globules in the sample cell are situated at different places ∼100 µm apart from each other, as shown in the schematics. The pictures were colored by the superposition of spectrographs. The bar is 5 µm. (B) The Japanese character “kyo” was constructed from nine rods of the DNA assembly over a total time of about 20 min. The complete character is also shown as a quasi-3D image of fluorescence. Scale bars are 10 µm. “Kyo” means “metropolis” but is in practice used to refer to the city of Kyoto.
sponding to the experimental results with the DNA/CholPEG-A complex (Figure 1C right). Thus, it is apparent
Conclusion In conclusion, we have demonstrated a new strategy for fabricating molecular assemblies from individual DNA molecules by using laser trapping. Since the fundamental principle is simple, we expect that a similar strategy can be applied to different kinds of macromolecules, both synthetic and natural, as well as colloidal particles. Consideration of the interacting potential between microor nanoparticles and external fields is the key to successful results. Acknowledgment. M.I. was supported by a JSPS Research Fellowship for Young Scientists. LA034338T
