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J. Phys. Chem. 1996, 100, 19702-19705
Intrachain Segregation in Single Giant DNA Molecules Induced by Poly(2-vinylpyrrolidone) 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 25, 1996; In Final Form: September 9, 1996X
Microsegregation in individual single chains of T4 DNA in solutions of poly(2-vinylpyrrolidone) (PVP) was observed by fluorescent microscopy. After mixing solutions of DNA with PVP, single DNA chains exhibited intrachain segregation with multiple mini-globules connected by narrow flexible chains. The microsegregated form of DNA was observed only in solutions of high-molecular-weight PVP (Mw ∼ 220 000), while in solutions of short chain PVP (Mw ∼ 10 000), only the randomly coiled conformation was found. The average lifetime of microsegregated DNA chains was more than several days while some of the microsegregated DNA molecules changed into a completely collapsed state.
Introduction Transition between random coil and compact globule states is an important property of isolated long polymer chains. Despite the theoretical prediction of the existence of a firstorder phase transition between the coil and globule states for isolated stiff polymer chains,1,2 almost all of the experimental studies to date have reported that this transition has a continuous or diffuse nature.3,4 Recently, we presented direct evidence of a discrete coil-globule transition in a single duplex DNA chain by using fluorescence microscopy.5-10 It has also been shown that the addition of poly(ethylene glycol) (PEG)6-8 results in a discrete coil-globule transition on individual DNA molecules. Thus, the transition between the elongated coiled state and the completely collapsed state is all-or-nothing at the level of individual DNA chains. On the other hand, it has been demonstrated that11 the transition appears continuous at the level of an ensemble of many chains. In the present paper, we describe the direct observation of single T4 DNA chains in aqueous solutions of high-molecularweight poly(2-vinylpyrrolidone) (PVP). We used fluorescence microscopy to study the conformational changes in single macromolecules. The dye 4′,6-diamidino-2-phenylindole (DAPI) was used to visualize DNA molecules. This method is widely used to study the dynamic and conformational changes12-16 of long DNA molecules, and its rationale has been described elsewhere.5-10 Experimental Section Bacteriophage T4dC DNA (166 kilobase pairs (kbp), contour length 55 µm14) was purchased from Nippon Gene. The fluorescent dye DAPI and the antioxidant 2-mercaptoethanol (ME) were obtained from Wako Pure Chemical Industries Ltd. PVP with Mw ) 10 000 and 220 000 was obtained from Tokyo Kasei Kogyo Co. A concentrated stock solution of 1:1 DNADAPI complex (DNA concentration in nucleotide 6 × 10-5 M) was added to a solution of PVP in 0.5 TBE buffer containing 4 vol % ME. The final concentration of DNA was 6 × 10-7 M. After gentle mixing, the solution was placed on a microscope slide and covered with a coverslip at a thickness of † Permanent address: Department of Polymer and Crystal Physics, Faculty of Physics, Moscow State University, Moscow 117234, Russia. * To whom all correspondence should be addressed. E-mail: F43943a@ nucc.cc.nagoya-u.ac.jp. FAX: +81-52-789-4808. X Abstract published in AdVance ACS Abstracts, November 1, 1996.
S0022-3654(96)01887-4 CCC: $12.00
∼100 µm. The solution was isolated from the air by nail enamel. The prepared samples were allowed to stand for 20 min, unless otherwise indicated, and observations were then carried out at room temperature (∼20 °C). Results and Discussion Figure 1a shows a typical fluorescence image of a T4 DNA chain, exhibiting translational and intramolecular Brownian motion in 0.5 TBE buffer solution without PVP. As a characteristic parameter to represent the dimension of individual polymeric chains, we used the apparent length of the long axis, Lmax, which was defined as the longest distance in the outline of the DNA image. The images were evaluated with an image processor (Argus 10, Hamamatsu Photonics) in individual video frames. For the ensemble of individual molecules, mean-square values of Lmax for at least 60-100 individual images were used. The images show a broad distribution of Lmax, as indicated in histogram a in Figure 2, which represents the three different morphologies: elongated coil, intrachain segregation, and compacted globule. The morphology of individual DNA chains was based on a time series of the video image. Since individual DNAs exhibit thermal intrachain motion, it is not difficult to classify individual chains. The addition of PVP to a solution of DNA containing DAPI produced a large increase in the intensity of the back ground fluorescence and, hence, less pronounced DNA images. This result can be explained by the formation of a complex between DAPI and PVP in competition with DAPI-DNA. Due to this effect, observations were almost impossible at concentrations of PVP greater than 25%. Figure 3 shows the dependence of Lmax on the concentration of PVP in the solution. The resulting conformational state of DNA depends strongly on the length of the PVP chain. An increase in the content of PVP with Mw ) 10 000 in the solution of DNA to a concentration of 15% produces essentially no change in the conformation of DNA chains, which remain in the coil state and display random intramolecular thermal fluctuation. The histogram of the Lmax values demonstrates a broad unimodal distribution similar to that obtained for buffer solution in the absence of PVP (Figure 2b). A further increase in the PVP concentration up to 25% induces slight additional swelling of the coils. Even at the highest concentration of PVP with Mw ) 10 000 (50%), none of the DNA chains show any bright globular structures. © 1996 American Chemical Society
Microsegregation in Single Chains of T4 DNA
J. Phys. Chem., Vol. 100, No. 50, 1996 19703
Figure 1. (A) Fluorescence images of T4DNA molecules moving freely in buffer solution. (a) Elongated coil in the 0.5*TBE buffer solution, (b) coexistence of coil and globule, in the presence of 4% PEG, (c) compacted globule with 15% PVP (Mw ) 220 000), and (d) intrachain segregation with 8.8% PVP (Mw ) 220 000). (B) Schematic representation of the change in DNA morphology with the addition of PEG and PVP. Both synthetic polymers induce a compacted globule at higher concentrations.
In solutions of PVP with Mw ) 220 000, single DNA chains transition to the collapsed state. At a PVP concentration of 1.8%, the coils shrink slightly. A further increase in the PVP concentration up to 3.4% results in a sharp decrease in Lmax, accompanied by the appearance of two or more bright spots along a single chain, as shown in Figure 1d. This result can be explained by using the concept of microsegregation in individual DNA chains, where two or more small globules are connected by flexible chains. Since microsegregated chains usually coexist with completely unfolded coiled chains in Figure 2c, the distribution of the Lmax values is somewhat wider. To distinguish between intrachain segregation and the aggregation of many chains, a series of experiments with very diluted DNA solutions was performed. In this case, concentrated PVP solution was mixed with diluted DNA solution (concentration of nucleotides 6 × 10-8 M; DAPI 2 × 10-8 M). At this concentration, DNA molecules are separated from each other by as much as a few tens of micrometers. However,
microsegregated chains were still observed. Microsegregated DNA chains were also observed in very diluted solutions of λ phage DNA, which is shorter than T4 DNA (λ DNASPCLN 48.5 kbp, contour length 16 µm, obtained from Nippon Gene). At a PVP concentration of 5.9-15%, DNA completely shrinks and collapsed DNA appears together with intrachain segregated DNAs (Figure 1c). At the same time, unfolded coils completely disappear. This trend corresponds to a further decrease in Lmax (Figure 3, histograms e and g in Figure 2). An essential feature of the state of long-chain DNA in the presence of PVP is evolution with time. The tendency of this evolution differs between low and high PVP concentrations. Open circles in Figure 3 represent the plot of Lmax vs PVP concentration 1 day after sample preparation at room temperature. Comparison of these data with those obtained for freshly prepared solutions indicates that, at a PVP concentration of 8-15%, the DNA chains undergo further folding. Analyses of the values of Lmax together with histograms g and h and i
19704 J. Phys. Chem., Vol. 100, No. 50, 1996
Starodoubtsev and Yoshikawa
Figure 3. Plot of the mean long-axis length of T4 DNA molecules vs the concentration of PVP with Mw ) 10 000 (closed triangle) and 220 000 (closed circle). The open circles indicate the values 1 day after sample preparation with PVP (Mw ) 220 000). The closed square corresponds to the measurement for the sample of 5 days after the dilution with the buffer solution.
Figure 2. Histograms of the distribution of T4 DNA long-axis lengths at various concentrations of PVP, with three different DNA morphologies: dotted, elongated coil; shaded, intrachain segregation; solid, globule; (a) 0.5*TBE buffer solution; (b) 15% PVP Mw ) 10 000; (cj) PVP Mw ) 220 000: (c, d) 3.65%, (e, f) 5.87%, (g, h) 8.80%, and (i, j) 14.75%; (d, h, j) the distribution of 24 h after sample preparation; (f) after 5 days. The other histograms show the results 20 min after sample preparation.
and j in Figure 2 show that the fraction of segregated coils decreases with time. For example, with a PVP concentration of 8.8%, there are 96% of microsegregated coils and 4% of globules immediately after the preparation, while these values are 32% and 68%, respectively, after 1 day. In contrast to the time-dependent change with a higher concentration of PVP, at lower PVP concentrations, Lmax tends to increase with time (Figure 3). To understand this effect, we must consider that, in solutions of PVP during sample preparation, a large fraction of segregated DNA is adsorbed on the surface of the microscope slide. Thus, the increase in the apparent dimensions of DNA molecules in the region of microsegregation may be explained by fractional adsorption of
segregated chains, which produces a relative increase in the population of coiled DNA in the solution. The reversibility of microsegregation was checked as follows. A DNA sample was prepared in 5.9% PVP and then allowed to stand for 20 min at room temperature. Under these conditions, more than 95% of the DNA chains undergo microsegregation. Next, the DNA solution was diluted 4-fold with buffer and studied after repeated immersion at room temperature for 5 days. After this treatment, segregated chains unfold and nearly all of the microglobules completely disappear. Even after several days of incubation with 5.9% PVP, DNA did not completely unfold and the microsegregated chains remained in the solution together with unfolded coiled chains. The comparison of the histograms for DNA from stock solution in buffer (Figure 2a) and those for DNA with completely unfolded chains after segregation in 5.9% PVP (Figure 2f) shows that the Lmax values are slightly different from each other. This trend may be attributed to the rupture of long DNA chains during the repeated mixing of DNA with highly viscous PVP solution and buffer. In conclusion, let us compare the present results with previously obtained results for T4 DNA in solutions of another neutral linear polymer, PEG (see Figure 1). In previous reports,6-8 direct evidence of a discrete coilglobule transition in a single duplex DNA induced by the addition of PEG was obtained using of fluorescence microscopy. It has been demonstrated that the transition of an unfolded single DNA coil into a globule is a first-order phase transition. The transitions of DNA single chains in the presence of PEG are schematically illustrated in Figure 1B. The results of this work demonstrate another route for compaction of single DNA coils. With PVP, the first step in DNA folding is the microsegregation of single chains with the formation of two or more small globules. At an intermediate concentration of PVP, this partly folded conformation is reasonably stable and further folding into a single globule proceeds very slowly. The difference in hydrophobicity between PEG and PVP may produce the different characteristics of the transition. In a hydrophobic environment with PVP, the negative charge of DNA is expected to be considerably reduced, causing a decrease in persistence length of the DNA chain. From theoretical perspective2 and recent experimental results,17 the discrete nature of the coil-globule transition will be diminished for a less-stiff polymer. Thus, the all-or-nothing nature of the transition will be modulated in a hydrophobic environment, such as PVP solution. Acknowledgment. S.S.G. is grateful to the Japan Society for the Promotion of Science, which provided the opportunity
Microsegregation in Single Chains of T4 DNA for his work in Nagoya University. This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture. References and Notes (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) Post, K. B.; Zimm, B. H. Biopolymers 1982, 21, 2123. (4) Murphy, L. D.; Zimmerman, S. V. Biophys. Chem. 1995, 57, 71. (5) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Matsumoto, M.; Doi, M. FEBS Lett. 1991, 295, 67. (6) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Khokhlov, A. R.; Doi, M. Biopolymers 1994, 34, 555. (7) Vasilevskaya, V. V.; Khokhlov, A. R.; Matsuzawa, Y.; Yoshikawa, K. J. Chem. Phys. 1995, 102, 6595. (8) Yoshikawa, K.; Matsuzawa; Y. Physica D 1995, 84, 220.
J. Phys. Chem., Vol. 100, No. 50, 1996 19705 (9) Yoshikawa, Y.; Yoshikawa, K. FEBS Lett. 1995, 361, 277. (10) Melnikov, S. M.; Sergeev, V. G.; Yoshikawa, K. J. Am. Chem. Soc. 1995, 117, 2401. (11) Yoshikawa, K.; Takahashi, M.; Khokhlov, A. R.; Vasilevskaya, V. V. Phys. ReV. Lett. 1996, 76, 3029. (12) Matsumoto, S.; Morikawa, K.; Yanagida, M. J. Mol. Biol. 1991, 152, 501. (13) Bustamante, C. Annu. ReV. Biophys. Biophys. Chem. 1991, 20, 415. (14) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K., Matsumoto, M.; Toshimasa, S.; Kimura, H.; Doi, M. J. Polym. Sci., Part B: Polym. Phys. 1992, 30, 779. (15) Minagawa, K.; Matsuzawa, Y.; Yoshikawa, K.; Masubushi, Y.; Matsumoto, M.; Doi, M.; Nishimura, C.; Maeda M. Nucleic Acids Res. 1993, 21, 37. (16) Yoshikawa, K.; Matsuzawa, Y.; Minagawa, K.; Doi, M.; Matsumoto, M. Biochem. Biophys. Res. Commun. 1992, 188, 1274. (17) Chu, B.; Ying, Q.; Grosberg, A. Yu. Macromolecules 1995, 28, 180.
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