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Compaction and Multiple Chain Assembly of DNA with the Cationic Polymer Poly(aluminum chloride) (PAC) Yukiko Matsuzawa,† Toshio Kanbe,‡ and Kenichi Yoshikawa*,§ Department of Ecological Engineering, Toyohashi University of Technology, Toyohashi, Aichi 441-8580, Japan, Laboratory of Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan, and Department of Physics, Kyoto University & CREST, Kyoto 606-8502, Japan Received December 18, 2003. In Final Form: March 19, 2004 Assembly of DNA molecules by the addition of poly(aluminum chloride) (PAC) was studied. In the absence of PAC, electron microscopy indicated the formation of elongated coiled DNA molecules. In the presence of PAC, multiple doughnut-like structures, 8-15 nm thick, formed and fused together. When salt was added, the doughnut-like structures tended to be thinner and the morphology of the fused doughnuts became irregular. We obtained a view of a single DNA structure by fluorescent microscopy, which revealed that individual DNA molecules undergo a discrete transition from an elongated to compacted state with an increase in PAC concentration. Electron microscopic observation showed that a regular doughnut is the typical structure seen under low salt conditions. At high salt concentrations, the doughnut shape deformed, yielding results similar to those produced by the salt effect on DNA assembly at high DNA concentrations.
Introduction DNA is a highly specific functional biopolymer and is one of the most important materials in biology as well as in polymer science. The solution behavior of DNA has been studied by various techniques, including electron microscopy,1 light scattering,2 sedimentation,3 circular dichroism,4 and fluorescent microscopy,5 which detect changes in size, chirality, or morphology of the DNA. The change in the higher order structure of DNA has been frequently interpreted in terms of DNA condensation. The term “DNA condensation” has been applied to the assembly phenomenon of DNA molecules without distinguishing single DNA compaction from multiple DNA aggregation. Previous reports have stated that 76% of the phosphate groups on a DNA strand are shielded by counterions in aqueous solution and that DNA condensation occurs at a negative shielding charge >89%.6,7 Some of the past studies on DNA condensation using sedimentation or light scattering at low DNA concentrations have suggested the possibility of a bimodal distribution of DNA conformations composed of expanded coils and condensation structures.3 On the other hand, DNA condensation at high DNA concentrations has been usually regarded as essentially the same as compaction of a single DNA molecule.4 The experimental technique of single molecular chain observation using fluorescence microscopy has indicated that individual large DNA molecules undergo an all-or-none transition from an elongated coil to a compacted globular * To whom correspondence should be addressed. E-mail:
[email protected]. † Toyohashi University of Technology. ‡ Nagoya University School of Medicine. § Kyoto University. (1) Hud, N. V.; Downing, K. H. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 14925. (2) He, S.; Arscott, P. G.; Bloomfield, V. A. Biopolymers 2000, 53, 329. (3) Lerman, L. S. Proc. Natl. Acad. Sci. U.S.A. 1971, 68, 1886. (4) Jordan, C. F.; Lerman, L. S.; Venable, J. H. Nature 1972, 236, 67. (5) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Doi, M.; Khokhlov, A. R. Biopolymers 1994, 34, 555. (6) Wilson, R. W.; Bloomfield, V. A. Biochemistry 1979, 18, 2192. (7) Manning, G. S. Q. Rev. Biophys. 1978, 11, 179.
state.8,9 This discrete nature of the transition is found to be rather general, occurring despite differences in the condensing chemicals, such as multivalent metal cations, polyamines, poly(ethylene glycol) (PEG)-salt, or alcohols.10-13 Toroidal and rod structures can accompany DNA condensation.14,15 The toroidal or rod-shaped objects are generated from multiple DNA molecules as well as from compaction of a single giant DNA molecule. In addition, DNA aggregation at high DNA concentrations has been investigated thoroughly as a model of the highly packed DNA found in cell nuclei.16,17 At high concentrations, DNA in ethanol mainly forms a fibrous network structure.18 Bacteriophage T4 DNA produces a concentric or tightly packed spiral,19 and DNA molecules in vivo in sperm heads form highly ordered cholesteric structures.20 Precipitation allows us to quantify ion condensation without the help of sophisticated local measurements. Hud et al. reported the image of a salmon protamine-DNA complex and sperm chromatin by atomic force microscopy (AFM), showing closely packed nodules 500-1000 Å in diameter.21 They proposed a toroidal structure for the fundamental packing unit of sperm DNA. Protamine is an arginine-rich protein that generates a toroid and rod structure. However, the ability of DNA to (8) Yoshikawa, K.; Matsuzawa, Y. J. Am. Chem. Soc. 1996, 118, 929. (9) Yoshikawa, K.; Matsuzawa, Y. Physica D 1995, 84, 220. (10) Yamasaki, Y.; Yoshikawa, K. J. Am. Chem. Soc. 1997, 119, 10573. (11) Takahashi, M.; Yoshikawa, K.; Vasilevskaya, V. V.; Khokhlov, A. R. J. Phys. Chem. B 1997, 101, 9396. (12) Makita, N.; Yoshikawa, K. Biophys. Chem. 2002, 99, 43. (13) Ueda, M.; Yoshikawa, K. Phys. Rev. Lett. 1996, 77, 2133. (14) Widom, J.; Baldwin, R. L. J. Mol. Biol. 1980, 144, 431. (15) Arscott, P. G.; Li, A. Z.; Bloomfield, V. A. Biopolymers 1990, 30, 619. (16) Reich, Z.; Wachtel, E. J.; Minsky, A. Science 1994, 264, 1460. (17) Pelta, J., Jr.; Durand, D.; Doucet, J.; Livolant, F. Biophys. J. 1996, 71, 48. (18) Richards, K. E.; Williams, R. C.; Calendar, R. J. Mol. Biol. 1973, 78, 255. (19) Lang, D. J. Mol. Biol. 1973, 78, 247. (20) Livolant, F. Physica A 1991, 176, 117. (21) Hud, N. V.; Allen, M. J.; Downing, K. H.; Lee, J.; Balhorn, R. Biochem. Biophys. Res. Commun. 1993, 193, 1347.
10.1021/la036392f CCC: $27.50 © 2004 American Chemical Society Published on Web 06/24/2004
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Figure 1. (A) Fluorescent images of T4 DNA at PAC concentrations of (a) 0.05, (b) 0.1, and (c) 0.5 ng/mL along with schematic representations of the relationship between the actual DNA chain conformation and the corresponding fluorescent image. (B) Distribution of the long axis length of DNA at 0.05, 0.07, 0.1, 0.2, and 0.5 ng/mL, based on analysis of 100 DNA molecules at each concentration.
form assembly/aggregate structures has not been fully understood yet. In this paper, we describe the adaptation of the popular cationic coagulant poly(aluminum chloride) (PAC) to explore interactions with DNA by results found at low and high DNA concentrations. Experimental Section Materials. DNA from salmon sperm (Mw, (2-3) × 106) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). T4 phage DNA (166 kbp, Nippon Gene, Toyama, Japan) was used for the direct observation of DNA by fluorescent microscopy. YOYO-1 [(1,1′-4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-oxazole)-2methylidene]-quinolinium tetraiodide] from Molecular Probes, Inc. (Eugene, OR) was used as a fluorescent dye. 2-ME (2mercaptoethanol) from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) was the antioxidant. PAC was a gift from NGK Ltd. (Nagoya, Japan).22 Formation of the DNA-PAC Complex on a Macroscopic Scale. Salmon sperm DNA was dissolved in distilled water at a concentration of 10 mg/mL and used as the stock DNA solution for DNA precipitation experiments. The stock concentration of PAC was 1 mg/mL. DNA and PAC were mixed to obtain the complex in 10 mM Tris-HCl buffer (pH 7.2). According to the colloid titration, the electric charge of [1 µg/mL PAC]/[1 mg/mL DNA] was 0.9.23 After vortexing, the sample was allowed to stand at room temperature for 2 h and then was centrifuged for 10 min at 10 000 rpm. A 100-µL sample of the supernatant was used to (22) Ban, S. Nikkakyo Geppo (in Japanese) 1973, 1, 27. (23) Ueno, K.; Kina, K. J. Chem. Educ. 1985, 62, 627.
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Figure 2. Electron micrograph of single compacted DNA molecules at a DNA concentration of 0.2 µg/mL: (A) DNAPAC complex without NaCl at a PAC concentration of 0.5 ng/ mL; (B) DNA-PAC complex at 0.3 M NaCl at a PAC concentration of 0.8 ng/mL. The scale bar is 50 nm. measure the absorbance at 260 nm with a BioSpec 1600 spectrophotometer (Shimadzu). Observation of Individual DNA Molecules by Fluorescent Microscopy. T4 DNA stock solution was dissolved in a Tris-HCl buffer (pH 7.2) and mixed with YOYO-1, 2-ME, and PAC. The final concentrations were as follows: 10 mM TrisHCl buffer, 0.2 µg/mL DNA in nucleotide, 0.03 µM YOYO-1, and 2% (v/v) 2-ME. The sample stood at room temperature for 2 h to achieve equilibrium. A 5-µL portion of the sample was transferred onto a glass slide by a pipet. The fluorescence image of the DNA molecule was obtained using an Olympus microscope, IX-70, equipped with a 100× oil-immersion objective lens and a highly sensitive Hamamatsu silicon intensified target (SIT) TV camera. Electron Microscope Measurements. Transmission electron microscopic observation of the complex between DNA and PAC was obtained using the negative staining method. A copper grid (no. 400) was coated with Formvar. The grid was floated on a droplet of sample solution (15 µL) for 3 min, then placed on filter paper, and then stained with an aqueous solution of 1% uranyl acetate for 10 s. The sample was observed with a JEOL 1200 EX transmission electron microscope (Tokyo, Japan) at 100 kV.
Results and Discussion Conformational Transition of Single DNA Molecules. Figure 1A shows fluorescent images of T4 DNA molecules at different PAC concentrations, indicating that DNA molecules undergo a transition from an elongated coil state to a folded compacted state. Figure 1B shows the distribution of the long axis length of T4 DNA at different concentrations of PAC in a diluted DNA solution at 0.2 µg/mL. The long axis length was observed by fluorescence microscopy as the longest distance in the
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Figure 4. (A) DNA precipitation with different concentrations of PAC at 0.3 M NaCl. The white arrow indicates the conditions under which the electron micrograph was obtained. (B) Electron micrograph of DNA and PAC complexes at 0.3 M NaCl as shown by the arrow in part A. The scale bar is 50 nm.
Figure 3. (A) Salmon sperm DNA precipitation at different concentrations of PAC, with DNA concentration kept at 1 mg/ mL. (B) Electron micrographs of DNA and PAC complexes (stained with 1% uranyl acetate) at PAC concentrations of (a) 0, (b) 1, (c) 2.5, (d), and (e) 5 µg/mL. The scale bar is 200 nm for parts a-d and 50 nm for part e.
outline of the DNA image. At low PAC concentrations (
