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Conformational Transition of Large Duplex T4 DNA Embedded in Poly(acrylamide) Gel S. G. Starodoubtsev† and K. Yoshikawa* Division of Informatics for Natural Sciences, Graduate School of Human Informatics, Nagoya University, Nagoya 464-01, Japan Received June 24, 1997. In Final Form: October 27, 1997X Changes in the conformation of duplex T4 DNAs embedded in poly(acrylamide) (PAAm) gels (as opposed to in an aqueous environment) were studied using fluorescence microscopy for the observation of individual molecules. In aqueous solutions, individual DNA chains exhibited thermal fluctuation, i.e., intramolecular and translational Brownian motion. With the addition of acetone, DNAs were adsorbed onto the surface of a glass plate accompanied by a discrete change in conformation from an elongated coil into a collapsed globule, while “metastable” coiled DNAs were present in the buffer solution with a lifetime of several days. In contrast, in the gel phase, embedded DNAs did not exhibit thermal motion. With the addition of acetone, elongated-coil DNA underwent a sharp but continuous transition driven by the collapsing transition of the gel. The resulting compact DNA was shrunk less than the collapsed DNA in free aqueous solution. In addition, it is shown that elongation of the PAAm gel affects the orientation of the DNA chains.
Introduction The transition between random coil and compact globule states is an important property of isolated long polymer chains.1,2 An analogous transition has been found in gels with weakly cross-linked chains of a polymer network. For polyelectrolyte gels, the coil-globule transition of the subchains is highly cooperative and usually proceeds as a first-order phase transition.3-5 On the other hand, almost all of the experimental studies to date have reported that the coil-globule transition has a continuous or diffuse nature.6-9 In the past experiments, there seems to have been almost no clear distinction between single-chain collapse and multichain condensation, due to the limitations of the available experimental methods.6,9 Recently, we presented direct evidence of a marked discrete coilglobule transition in isolated duplex DNA chains using fluorescence microscopy to achieve single-chain observation.10-16 It has been shown that the addition of * To whom correspondence should be addressed: e-mail,
[email protected]; tel, +81-52-789-4849; Fax, +8152-789-4808. † Permanent address: Department of Polymer and Crystal Physics, Faculty of Physics, Moscow State University, Moscow 117234, Russia. X Abstract published in Advance ACS Abstracts, December 15, 1997. (1) Lifshits, I. M.; Grosberg, A. Yu.; Khokhlov, A. R. Rev. Mod. Phys. 1978, 50, 683. (2) Grosberg, A. Yu.; Khokhlov, A. R. Statistical Physics of Macromolecules; American Institute of Physics Press: New York, 1994. (3) Tanaka, T. Phys. Rev. Lett. 1978, 40, 820. (4) Tanaka, T.; Fillmore, D.; Sun, S. T.; Nishino, J.; Swislow, G.; Shah, A. Phys. Rev. Lett. 1980, 45, 1636. (5) Khokhlov, A. R.; Starodoubtsev, S. G.; Vasilevskaya, V. V. Adv. Polym. Sci. 1993, 109, 123. (6) Post, K. B.; Zimm, B. H. Biopolymers 1982, 21, 2123. (7) Murphy, L. D.; Zimmerman, S. V. Biophys. Chem. 1995, 57, 71. (8) Geiduschek, E. P.; Gray, I. J. Am. Chem. Soc. 1956, 78, 879. (9) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334. (10) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K., Matsumoto, M., Doi, M. FEBS Lett. 1991, 295, 67. (11) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Khokhlov, A. R.; Doi, M. Biopolymers 1994, 34, 555. (12) Vasilevskaya, V. V.; Khokhlov, A. R.; Matsuzawa, Y.; Yoshikawa, K. J. Chem. Phys. 1995, 102, 6595. (13) Yoshikawa, K.; Matsuzawa; Y. Physica D 1995, 84, 220. (14) Yoshikawa, Y.; Yoshikawa, K. FEBS Lett. 1995, 361, 277.
polyethylene glycol10-13 and other condensing agents14-16 results in a discrete coil-globule transition in a single DNA molecule. Thus, the transition between a random elongated coil and a compact globule is all-or-none at the level of individual DNA chains; i.e., this is a first-order phase transition. In a previous paper,17 we described a new microsegregated form, or intrachain phase separation, among individual chains of T4 and Lambda DNA in the presence of the flexible polymer poly(2-vinylpyrrolidone) (PVP). In the presence of PVP with a high enough molecular weight (Mw ∼ 220 000), single DNA molecules exhibit intrachain segregation, with multiple miniglobules connected by flexible coiled chains. It has also been shown that such a segregated form in DNA is metastable and slowly transforms into a completely collapsed globule state. In the present paper, we will compare the effect of acetone on the conformational behavior of long duplex DNA molecules in aqueous solutions with that in a poly(acrylamide) (PAAm) gel. The purpose of the present study is (i) to find the conditions to entrap long DNA without chemical damage, (ii) to check the possibility of controlling the conformation of long DNA molecules with the regulation of the macroscopic structure of the gel, and (iii) to know the effect of reptation as the relaxation process of long DNAs within the gel. Our answers on the above problems as the result of the present research are (i) long DNAs can be trapped within the gel without breaking of the chains, (ii) the conformation of individual DNA chains is controlled by the macroscopic change of the gel structure, and (iii) the conformation is frozen in the gel without reptation. We used fluorescence microscopy to study the conformational changes in isolated DNA macromolecules in an extremely dilute solution of DNA (less than 0.1 µm in base pairs), to avoid the effect of the interaction between DNA chains. This method is widely used to study the dynamic and conformational changes18-22 of long DNA (15) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401. (16) Yoshikawa, K.; Takahashi, M.; Vasilevskaya, V. V.; Khokhlov, A. R. Phys. Rev. Lett. 1996, 76, 3029. (17) Starodoubtsev, S. G.; Yoshikawa, K. J. Phys. Chem. 1996, 100, 19702.
S0743-7463(97)00668-9 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/06/1998
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molecules, and its rationale has been described elsewhere.10-17,23 Experimental Section Bacteriophage T4dC DNA (166 kilobase pairs (kbp), contour length 57 µm23) was purchased from Nippon Gene. DAPI and the antioxidant 2-mercaptoethanol (ME) were obtained from Wako Pure Chemical Industries Ltd. A standard 0.5xTBE buffer solution (45 mM Tris, 45 mM borate, 1 mM EDTA, pH 8.3 in water) was used. To prepare DNA solutions, a concentrated stock solution of 1:1 DNA-DAPI complex (DNA concentration in bp 6 × 10-5 M) was added to the aqueous TBE solution containing 4 vol % of ME. Acetone was then added to the DNA solution. A preliminary examination showed that ME strongly inhibits the polymerization of acrylamide (AAm). It was also noted that DAPI readily decomposes during polymerization reactions. To avoid these effects, we adapted the following procedure to prepare a gel for entrapping DNAs. DNA was immobilized in PAAm gel during copolymerization of AAm with N,N′-methylenebis(acrylamide) (200:1 mol; total concentration 6 wt %) in 0.5xTBE buffer containing T4 DNA (concentration, 6 × 10-7 M in bp). Ten milliliters of the initial mixture for gel formation was heated to 34 °C, and to this was added 5 µL of N,N,N,N-tetramethylethylenediamine and 50 µL of 10% ammonium persulfate solution. After gentle mixing, the solution was placed on a microscope slide and covered with a glass plate at a thickness of ∼100 µm. The solution was isolated from the air by nail enamel. The prepared samples were allowed to stand for 24 h at room temperature (∼20 °C). After polymerization was complete, the coverslip was taken off and the flat gel sample was removed from the surface of the microscope slide. It was then cut into centimeter pieces, and the pieces were immersed in water or water-acetone solutions in a refrigerator for 24 h at +4 °C in 0.5xTBE buffer containing 4 vol % of ME and 6 × 10-7 M DAPI. The sample solutions were then allowed to stand at room temperature for 4 h. The resulting flat samples were put on a microscope slide and dipped in a layer of buffer solution. The excess solution was removed using filter paper. Finally, the gel samples were covered by a coverslip glass and shielded by nail enamel. The observations were then carried out at room temperature (around 20 °C). The immobilization of DNA in PAAm gel containing DAPI results in a strong increase in the background intensity of the fluorescence (especially for elongated gel) in comparison with DNA solutions and thus gives less-pronounced DNA images. This increase in the background level is attributable partly to the strong light-scattering in the gel phase and to the formation of a weak complex between DAPI and PAAm in competition with the formation of a DNA-DAPI complex. Special care was taken to clean the glass plates thoroughly before the fluorescence microscopy observations. They were soaked in hydrogen peroxide for more than 1 h, washed repeatedly with distilled water, immersed in ethanol for at least 1 h and washed twice in Millipore water. Finally, they were dried at 35 °C for 1 h. Fluorescence images of DNA molecules were observed using a Zeiss Axiovert 135TV microscope equipped with a 100× oilimmersed objective lens and were recorded on videotape using a high-sensitivity Hamamatsu SIT TV camera. The observations were carried out at room temperature, ca. 20 °C.
Results and Discussion Collapse of “Free” DNA Chains in Water-Acetone Solutions. Figure 1A(a) shows a typical fluorescence (18) Matsumoto, S.; Morikawa, K.; Yanagida, M. J. Mol. Biol. 1981, 152, 501. (19) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Matsumoto, M.; Toshimasa, S.; Kimura, H.; Doi, M. J. Polym. Sci., Part B: Polym. Phys. 1992, 30, 779-783. (20) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Masubushi, Y.; Matsumoto, M.; Doi, M.; Nishimura, C.; Maeda, M. Nucleic Acids Res. 1993, 21, 37. (21) Yoshikawa, K.; Matsuzawa, Y.; Minagawa, K.; Doi, M.; Matsumoto, M. Biochem. Biophys. Res. Commun. 1992, 188, 1274. (22) Bustamante, C. Annu. Rev. Biophys. Chem. 1991, 20, 415. (23) Matsuzawa, Y.; Yoshikawa, K. Nucleosides Nucleotides 1994, 13, 1415.
Figure 1. (A) Example of fluorescence images of T4 DNA molecules: (a) elongated coil; (b) compact globule attached to the glass surface, which originated from a single chain in 50 vol % acetone; (c) associated globules with multiple DNAs; (d) single DNA chain trapped in collapsed PAAm gel with 50 vol % acetone. (B) Schematic representation of different conformational states of T4 DNA corresponding to the each picture in (A).
Figure 2. Dependence of L of T4 DNAs present in the bulk solution on the volume fraction of acetone. In the metastable region above 40 vol % acetone, coiled DNAs (open squares) are present in a transient state with a lifetime of a few days and globular DNAs (closed circles) are observed only at the glass surface. The number of attached globular DNAs gradually increases over several days.
image (and a schematic representation in Figure 1B(a)) of an elongated-coil T4 DNA chain which exhibits translational and intramolecular Brownian motion in 0.5xTBE buffer solution. To characterize the size of the DNA coils, the apparent length of the long axis L was defined as the longest distance in the outline of the DNA image. Meansquare values of L for at least 50-100 individual images were obtained. Analogous images were obtained for DNA immobilized in PAAm gel, but these showed much less contrast. Images of single globules adsorbed on a glass surface with the addition of 50 vol % acetone are shown in Figure 1A(b). Due to a blurring effect,11 the apparent globule image (about 0.6 µm) is larger than the actual dimensions. In fact, the actual size of a single globule calculated from its diffusion coefficient has been found to be on the order of 0.1 µm.15 Figure 2 shows the dependence of the long-axis length L on the volume fraction of acetone in 0.5xTBE buffer. With up to 40% acetone, the DNA molecules are in the elongated coil state. Above 40% acetone, DNA chains tend to collapse into globules on the glass surface, whereas some fraction of DNAs remain as elongated coils, as a metastable state. At 55 vol % acetone, although considerable fraction of coil is observed just after the sample preparation, after several days most of the DNAs are adsorbed on the glass surface exhibiting the collapsed
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globule state, while a minor fraction shows the coiled state together with the existence of small number of the associations between multiple DNAs. In contrast to a previous study on the collapse of DNA induced by 2-propanol and other condensation agents,24 with acetone, globules are observed only on the surface of microscope slides and not in the solution. This result indicates that in water-acetone media, the adsorption of globules onto a glass surface is much stronger than that in water-alcohol mixtures. This can be explained by the difference in the chemical nature of the organic solvents; acetone is an aprotonic solvent while 2-propanol exhibits hydrogen bonding with other alcohol molecules and with water. Another characteristic feature of DNA collapse with the addition of acetone is the formation of associations, or precipitates, of multiple globules on the glass surface (Figure 1A(c)). Despite rather intensive observations, we have not yet succeeded in observing the time-dependent process on the transition of DNA from the coil state to the collapsed form. Recently, direct observation of the collapse transition of single DNA chains in solutions of polyethylene glycol has shown the existence of the process of nucleation and growth along a single T4 DNA chain.13,25 Nucleation is the ratedetermining step for the transition from a coil to a globule, and the lifetime of the metastable coil is on the order of 1 h. The probability of nuclear formation is highest at the end of the DNA chain. After the nucleus reaches a critical size, the remaining part of the chain is pulled into a collapsed nucleus. The long lifetime of the metastable state together with the characteristic kinetic behavior can be described within the framework of a first-order phase transition in a single chain. By analogy, a similar mechanism of polymer chain folding has been suggested for the collapse of polystyrene in cyclohexane, based on light-scattering measurements.26 When we observed the DNAs under the flux of a sample fluid between slides induced by gentle mechanical agitation, some of the adsorbed globules exhibited long thin flexible “tails” (data not shown). Apparently, this conformational state of DNA represents on intermediate phase-separated state, in which the globule and coil states occupy different parts of the same chain, mediated by interaction with the glass surface. At 55-70 vol % acetone, the conformation of DNA strongly depends on the history of the sample preparation. If acetone is added to the solution of DNA, DNAs tend to remain in solution for a rather long time, on the order of a day, and are not adsorbed on the glass surface. The precipitation is enhanced only at 80 vol % acetone. When the stock solution of DNA is added to a water-acetone solution containing the buffer, a larger fraction of the DNA precipitates on the surface, forming large aggregates, where the structure of the precipitate differs markedly from the structure of a single globule or associations of globules. Such large aggregates are attributable to a kind of liquid crystalline state27-29 Further study is needed to clarify the details of this structure. Regardless of the history of dependence on the formation of the adsorbed globule, the coil conformation of DNA chains observed in solution is metastable above 40 vol % acetone. Despite this thermodynamic instability, the (24) Yoshikawa, K.; Ueda, M. Phys. Rev. Lett. 1996, 77, 2133. (25) Yoshikawa, K.; Matsuzawa, Y. J. Am. Chem. Soc. 1996, 118, 929. (26) Chu, B.; Ying, Q. Macromolecules 1995, 28, 180-189. (27) Livolant, F. J. Phys. 1986, 47, 1605. (28) Rill, R. L.; Strzelecta, T. E.; Davidson, M. W.; Winkle, D. H. Physica A 1991, 176, 87. (29) Pelta, J.; Durand, D.; Doucet, J.; Livolant, F. Biophys. J. 1996, 71, 48.
Starodoubtsev and Yoshikawa
Figure 3. Histograms of L of single DNA coils in water (a) and water-acetone solutions with 40 (b) and 70 (c) vol % of acetone.
lifetime of the metastable coil state of DNA chains in water-acetone solutions is on the order of a few days, which suggests the existence of a relatively high activation barrier between the coil and globule states. This conclusion is in good agreement with the results of a recent study of the properties associated with a first-order phase transition in single DNA13,25 An interesting feature of the coil state of DNA in water-acetone mixtures is that the size of the coils is almost independent of the acetone content. Despite a large decrease in the dielectric constant and the quality of the solvent, the shape and width of the distribution of L remain essentially constant (Figure 3). A very weak dependence of L on the concentration of the neutral condensing agents polyethylene glycol and 2-propanol in coiled DNA up to the region of the coil-globule transition has also been observed.11-13,24 The weak dependence of L is expected to have a neat relationship on the large discrete character in the coil-globule transition. Further detailed studies, including the effect of counterion binding and also condensation,30,31 are necessary to fully understand this phenomenon. Collapse of Single Coils of T4 DNA Immobilized in PAAm Gel. The collapse of neutral and weakly charged PAAm gels in mixtures of water and acetone has been known ever since the pioneering work of Tanaka and coworkers.3,4 Figure 4 (curve 1) shows the semilogarithmic plot of the swelling ratio m/m0 of a PAA gel versus the vol % of acetone in mixtures with water in the presence of 0.5xTBE buffer. In this figure, m0 and m are the steric volumes of samples in equilibrium with aqueous buffer solution and water-acetone buffer solution, respectively. The resulting curve, which resembles that of a cooperative transition, is typical for the collapse of neutral PAAm gels in mixtures of water with various organic solvents.32 At a low acetone content, the mass of the gel decreases slowly, whereas a sharp but continuous transition is recognized above 30% acetone. Despite a significant volume change, the gel remains transparent below 50 vol % acetone. At 50 vol % acetone, the shrunken gel becomes turbid within (30) Oosawa, F. Polyelectrolytes; Marcel Dekker: New York, 1971. (31) Manning, G. S. Q. Rev. Biophys. 1978, 11, 179. (32) Starodoubtsev, S. G.; Khokhlov, A. R.; Vasilevskaya, V. V. Dokl. Akad. Nauk SSSR (Dokl. Phys. Chem.) 1985, 282, 392-395.
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Figure 4. Semilogarithmic plot of the swelling ratio m/m0 (1, closed square) of PAAm gel and V/V0 (2, open circle) of immobilized single DNA coils vs the volume fraction of acetone. The dashed line (3, triangle) represents the corresponding dependence for single DNAs in water-acetone solutions without gel, where the coil in the bulk solution and the globule absorbed on the surface are indicated by open and closed triangles, respectively (see Figure 2).
24 h. However, it becomes transparent again after 3 days due to structural rearrangement. Gel samples in solutions of more than 50% acetone remain turbid, perhaps due to something like a glass transition.33 Fluorescence microscopy measurements demonstrate that despite radical attack during gel preparation, individual elongated DNA coils can be immobilized and observed in the gel phase. The DNA coils are completely immobilized, i.e., no dynamic change or thermal fluctuation is observed. This result may be due to the small contour length of the chains of the PAAm network (∼300Å) in contrast to the large length of the Kuhn segment of duplex DNA (∼1000 Å). Detailed analyses based on the values determined for the length of the Kuhn segment of PAAm hydrogel with approximately the same cross-linked density (about ten monomer units) show that the distance between the ends of the subchains of the network in this case is about 50-70 Å. Thus, the possible remaining freedom of the DNA segments displaced due to Brownian movement is on the order of 101-102 Å, i.e., much less than the length of the Kuhn segment of DNA. An increase in the acetone concentration and gel shrinkage results in a decrease in the characteristic size of the DNA images. The histograms given in Figure 5 show the distribution of L in mixtures with different acetone contents. The distributions markedly shift to the region of smaller values of L with an increase in the acetone content. The relative change in the average volume occupied by a single DNA coil in the gel phase (V/V0) during the collapse of the PAAm network can be characterized by the change in (L/L0)3 ∼ V/V0, where L0 and L correspond to the longest distance in the outline of the DNA images obtained from coils in the gel in aqueous and water-acetone buffer solutions, respectively. Figure 4 (curve 2) shows the semilogarithmic plot of V/V0 vs the volume fraction of acetone in solution. Comparison of the relative changes in V/V0 and m/m0 (which characterize the dependence of the gel volume) clearly indicates that the conformation of the DNA chains follows the conformational state of the network. In mixtures of water and acetone, gel collapse results in the transition of unfolded coils in shrunken conformations. The shrunken coils in the collapsed gel are much less bright than the globules in solution (Figure 1d). Thus, by using immobilization in PAAm gels, it becomes clear that DNAs change in size in a continuous manner, which is not observed in solution. For T4 DNA in collapsed PAAm gel, L is about 1.0 µm, which is larger than that for collapsed DNA in acetone (33) Philippova, O. E.; Pieper, T. J.; Sitnikova, N. L.; Starodoubtsev, S. G.; Khokhlov, A. R.; Kilian, H. G. Macromolecules 1995, 28, 39953929.
Figure 5. Histograms of L of single DNA chains immobilized in a PAAm gel at various volume fractions of acetone: (a) aqueous TBE buffer; (b)-(f), vol % ) 10, 20, 30, 40, and 50, respectively.
Figure 6. Histograms of L of single DNA coils immobilized in elongated (shaded bar) and isotropic (open bar) PAAm gel, in the TBE buffer solution.
without gel (see Figure 1A(b)). The larger size of the collapsed DNA in the gel is attributable to the effect of the entanglement with the gel matrix, while the collapsed product of long DNA without gel can form tightly packed “crystal structures” such as toroid and rod.35 As for the conformation of orientated single DNA chains due to the elongation of the gel, we could not show the actual picture because of the poor contrast in the images of DNA in deformed gel. Figure 6 shows a histogram of L of DNA in an oriented gel sample (degree of elongation ∼200-250%), compared with that for an isotropic gel sample equilibrated with aqueous buffer solution. Inspection of the histograms shows that elongation of the gel is accompanied by an increase in L in approximately the same ratio. Acknowledgment. S.S.G. is grateful to the Japan Society for the Promotion of Science, which provided the opportunity for his work at Nagoya University. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by the Asahi Glass Foundation. LA9706680 (34) Alexandrova, T. A.; Vasserman, A. M.; Starodoubtsev, S. G. Vysokomolek. Soed. (Polym. Sci., USSR) 1985, 27B, 780-784. (35) Noguchi, H.; Saito, S.; Kidoaki, S.; Yoshikawa, K. Chem. Phys. Lett. 1996, 261, 527-533.
