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Thermo-Switching of the Conformation of Genomic DNA in Solutions of Poly(N-isopropylacrylamide) )
Ning Chen,†, Anatoly A. Zinchenko,*,†, Satoru Kidoaki,‡ Shizuaki Murata,† and Kenichi Yoshikawa§, † Graduate School of Environmental Studies, Nagoya University, Chikusa, Nagoya 464-8601, Japan, Institute for Materials Chemistry and Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan, §Graduate School of Science, Kyoto University, Sakyo, Kyoto 608-8501, Japan, and Spatio-Temporal Order Project, ICORP, JST, Tokyo, Japan )
‡
Received November 18, 2009. Revised Manuscript Received January 11, 2010 We used poly(N-isopropylacrylamide) (PNIPAM) to control the conformation of genomic DNA by changing the temperature of a reaction solution and studied the DNA transition at the level of single DNA molecules. With this method, the conformation of long genomic DNA can be readily and reversibly switched between a very compact condensate and an unfolded macromolecule.
Introduction
Experimental Section
The conformation of deoxyribonucleic acid (DNA) is closely related to the mobility of DNA in vivo and to the accessibility of the genetic information in DNA.1 The genetic activity of DNA, such as transcriptional activity, has been shown to correlate with its higher-order structure.2-4 Therefore, control of the higher-order structure of DNA has been widely investigated, especially in relation to the application of DNA for gene-delivery purposes. The higher-order structure of DNA can be readily controlled by adding various cationic binders that induce DNA charge-neutralization and compaction.5 In contrast, remote control of the DNA conformation by external stimuli is still a challenge. One promising approach is the use of a thermosensitive DNA binder, which can influence DNA higher-order structure and be reversibly bound or released with a change in temperature. Poly(N-isopropylacrylamide) (PNIPAM),6 which was first synthesized in 1950, has been shown to be useful in many applications, since an increase in temperature above 30 °C triggers a phase transition of the PNIPAM polymer, such as for DNA affinity separation,7,8 temperaturecontrolled self-assembly of gene delivery vectors,9 DNA sensors,10 and so forth. In this study, we used PNIPAM as an environmental chemical factor to reversibly control the conformation of genome DNA chains with changes in temperature, and observed a change in the structure of DNA at the level of single DNA molecules. *Corresponding author. E-mail:
[email protected].
(1) Mohd-Sarip, A.; Verrijzer, C. P. Science 2004, 306, 1484–1485. (2) Yamada, A.; Kubo, K.; Nakai, T.; Yoshikawa, K.; Tsumoto, K. Appl. Phys. Lett. 2005, 86–88. (3) Akitaya, T.; Tsumoto, K.; Yamada, A.; Makita, N.; Kubo, K.; Yoshikawa, K. Biomacromolecules 2003, 4, 1121–1125. (4) Zinchenko, A. A.; Luckel, F.; Yoshikawa, K. Biophys. J. 2007, 92, 1318– 1325. (5) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334–341. (6) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163–249. (7) Maeda, M.; Nishimura, C.; Inenaga, A.; Takagi, M. React. Polym. 1993, 21, 27–35. (8) Umeno, D.; Kawasaki, M.; Maeda, M. Bioconjugate Chem. 1998, 9, 719–724. (9) Oupicky, D.; Reschel, T.; Konak, C.; Oupicka, L. Macromolecules 2003, 36, 6863–6872. (10) Demirel, G.; Rzaev, Z.; Patir, S.; Piskin, E. J. Nanosci. Nanotechnol. 2009, 9, 1865–1871.
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Materials. T4 DNA was purchased from Nippon Gene Co. Ltd., Japan. PNIPAM (Mw = 20 000-25 000) was purchased from Aldrich, USA. The fluorescent dye DAPI (40 ,6-diamidino2-phenylindole) and NaCl were from Nacalai Tesque Inc. Deionized water (Milli-Q, Millipore) was used for all experiments. Fluorescence Microscopy. Fluorescence microscopy (FM) sample solutions were prepared by successive mixing of water, fluorescent dye DAPI (10-7 M), T4 DNA (10-7 M), and PNIPAM at different concentrations. Fluorescent microscopic observations were performed with a fluorescence microscope (Nikon, TE2000-E) equipped with a 100 oil-immersion lens. Fluorescent images were recorded using an EB-CCD camera and an Argus 10 image processor (Hamamatsu Photonics, Hamamatsu, Japan). For experiments that required temperature control, a thermoplate (Tokai Hit Company) was used. Transmission Electron Microscopy (TEM). Sample solutions for TEM were prepared in the same manner as for FM observations. TEM observations were performed at room temperature using an H-800 microscope (Hitachi) at an acceleration voltage of 150-200 kV. We used collodion-coated grids with a mesh size of 300 in the observations.
Results and Discussion The conformational dynamics of single DNA molecules (bacteriophage T4, 166 kbp) was monitored by fluorescence microscopy. First, DNA labeled with a fluorescent dye in aqueous solutions was mixed with PNIPAM (Mw = 20 000-25 000) at different ratios and the conformation of DNA was monitored. Single-molecule DNA fluorescence profiles in Figure 1A show that, with an increase in the PNIPAM concentration, the DNA conformation gradually changes from an unfolded coil (0% PNIPAM) to a compact globule (0.05% PNIPAM). Figure 1B shows the results of a statistical analysis of the average long-axis length of DNA: the long-axis length of DNA decreases from about 4 μm to about 1 μm. The formation of a DNA globule by ionic interaction between PNIPAM and DNA is unlikely, since there are no positively charged groups in the PNIPAM molecule. Thus, the mechanism of DNA compaction by PNIPAM should be much different from that with polycationic condensing agents. Therefore, the
Published on Web 01/27/2010
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Figure 1. DNA compaction in solutions of PNIPAM. (A) Typical fluorescence microscopy images of single DNA molecules (10-7 M) in aqueous solution at different concentrations of PNIPAM. (B) Dependence of the average DNA long-axis length on the increase in the PNIPAM concentration in aqueous solution.
two following mechanisms can be considered: weak binding of PNIPAM to DNA similarly to earlier studied DNA-polyamide systems,11 and the compaction of DNA in a crowding environment of PNIPAM. To clarify the mechanism, we studied the influence of a monovalent electrolyte (NaCl) on the compaction of DNA. The dependence of DNA average long-axis length on NaCl concentration in solution of 0.05% of PNIPAM is shown in Figure 2. The addition of NaCl at a concentration above ca. 10 mM induced gradual unfolding of the compacted DNA in PNIPAM solution (0.05%) from the globule state into the coil state. This unfolding is reversible and the further addition of a new portion of PNIPAM at the concentration 0.1% to the solution of unfolded DNA leads to recompaction of DNA into globules (Figure 2**). It is known that the effect of a monovalent electrolyte on the stability of a compact DNA complex differs according to the mechanism of DNA condensation. For example, DNA compaction in a crowding environment with a hydrophilic polymer such as poly(ethylene glycol) PEG is promoted by the addition of monocations;12 in contrast, the compaction of DNA by multications is inhibited by the addition of competing monocations.13 The observed effect of NaCl addition is similar to the effect of a multication in the compaction of DNA. However, other experiments indicate a contribution of crowding effect into DNA compaction in PNIPAM solution similarly to DNA compaction in solutions of neutral polymers such as poly(ethylene glycol) (PEG).12,14,15 When in sample containing DNA and PNIPAM of 0.015%, which corresponds to the intermediate stage of DNA compaction, a concentrated solution of PEG (polymerization degree ca. 200) was added until 7.5% concentration, all DNA molecules were found in the globule state indicating that PEG facilitated the compaction of DNA into globules, and thus, the contribution of depletion effect into DNA compaction in PNIPAM solutions was confirmed. In literature, it has been reported that, in order to compact T4 DNA in 1% solution of (11) Dervan, P. B.; Burli, R. W. Current Opin. Chem. Biol. 1999, 3, 688–693. (12) Evdokimov, Yu. M.; Akimenko, N. M.; Glukhova, N. E.; Varshavskii, Ya. M. Molekul. Biol. 1974, 8, 396–405. (13) Hibino, K.; Yoshikawa, Y.; Murata, S.; Saito, T.; Zinchenko, A. A.; Yoshikawa, K. Chem. Phys. Lett. 2006, 426, 405–409. (14) Vasilevskaya, V. V.; Khokhlov, A. R.; Matsuzawa, Y.; Yoshikawa, K. J. Chem. Phys. 1995, 102(16), 6595–6602. (15) Zinchenko, A. A.; Yoshikawa, K. Biophys. J. 2005, 88(6), 4118–4123.
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Figure 2. DNA decompaction in solutions of PNIPAM upon addition of NaCl and reentrant compaction by excess of PNIPAM. Dependence of the average DNA long-axis length on the increase in the NaCl concentration in solution of 0.1% of PNIPAM (open circles). Dependence of the average DNA long-axis length on the addition of 0.1% (*) and 0.2% (**) of PNIPAM solution into solution of unfolded DNA containing 0.1% of PNIPAM and 43 mM of NaCl (filled circles).
PEG of similar size to PNIPAM (ca. 200 monomer units) used in this study, the presence of more than 1 M NaCl is required14 to provide necessary charge neutralization of DNA. In contrast, in solutions of only 0.05% PNIPAM with no NaCl added DNA was compacted in this study, which indicates that DNA compaction cannot be explained solely due to the depletion effect. The above experiments suggest the dual role of PNIPAM in DNA compaction: PNIPAM macromolecules provide a crowding environment in solution and binding of PNIPAM to DNA provides necessary DNA charge neutralization. Therefore, the inhibiting effect of NaCl in DNA compaction in solutions of PNIPAM is, on one hand, attributed to electrostatic screening of weak interaction between DNA and PNIPAM and, on the other hand, probably due to the effect of sodium chloride on the diminishing of the excluded volume of PNIPAM, because the addition of salt to PNIPAM is known to induce contraction of the PNIPAM polymer.16 Next, we studied the conformational behavior of DNA in solution of PNIPAM at different temperatures. Heating of DNA in the coiled sate at concentrations of PNIPAM of less than 0.05% caused no changes in the DNA conformation. When a solution of compacted DNA globules containing more than 0.05% PNIPAM was heated, DNA underwent dramatic changes in conformation. Figure 3A shows the change in the DNA conformational state with a temperature increase and further chilling. An increase in temperature from room temperature (25 °C) to 30 °C had no effect on the DNA conformation. However, beginning at 30 °C DNA globules unfolded into a coil state. A further increase in temperature after DNA unfolding (above 36 °C) did not affect the conformation of the observed DNA macromolecules. Figure 3B also shows the results of a statistical analysis of the average DNA long-axis length upon heating and chilling, respectively. As shown, DNA is continuously decompacted from a compact globule into a DNA coil in the temperature range between 32 and 36 °C. When the temperature was gradually decreased from 36 °C back down to room temperature (Figure 3A,B), DNA coils were observed to undergo a re-entrance transition into the initial globules in exactly the same temperature range at which the decompaction of DNA took place during heating. A repeated increase in the temperature of the same sample causes DNA unfolding again, and it is expected that this heating-chilling cycle (16) Ikehata, A.; Ushiki, H. Polymer 2002, 43, 2089–2094.
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Figure 4. TEM images of DNA-PNIPAM complexes obtained by mixing DNA (10-7 M) and PNIPAM (0.05%) in water solution before (A-C) and after (D,E) a heating-cooling cycle. C and E are enlarged images of the middle part of the complexes shown in B and D, respectively.
Figure 3. Thermal switching of the DNA conformation. (A) Typical fluorescence microscopy images of a single DNA molecule (10-7 M) in a solution of 0.05% PNIPAM and in aqueous solution as a function of the solution temperature during heating (upper row) and chilling (bottom row). (B) Temperature-dependent change in the average DNA long-axis length during heating (open) and chilling (filled circles). (C) Temperature-dependent change in the UV absorbance of a solution identical to that used for FM observations at λ = 400 nm during heating (filled circles) and chilling (open circles).
can be performed many times, while the transition of DNA between the coiled and globule states can be reversibly controlled within a temperature range of 32-36 °C . It is well-documented that PNIPAM undergoes a marked conformation-based change in solubility at ca. 30 °C. To directly compare the point at which PNIPAM undergoes this change in conformation with the temperature of the DNA conformational transition, we recorded UV spectra of samples that were identical to those used in the microscopic experiments at different temperatures. An increase in temperature causes an increase in the optical absorbance of visible light, which corresponds to the coil-globule transition of PNIPAM, during which an insoluble phase of PNIPAM polymer is formed above the lower critical solution temperature (LCST). Figure 3C shows that the absorbance at λ = 400 nm begins to increase from 33 °C and reaches a plateau at 36 °C. A decrease in the solution temperature after heating causes the absorbance to decrease back to the initial value and the chilling curve corresponds exactly to the heating curve. Comparison of the changes in the UV spectra (Figure 3C) with those in the DNA conformation (Figure 3B) observed by FM reveals a clear correlation between the PNIPAM temperaturedependent conformation transition and DNA coil-globule compaction-decompaction, which is reversible and takes place at the same temperature upon heating or cooling. Due to the reversibility of the DNA conformational transition in PNIPAM solutions, the heating-cooling cycle can be performed repeatedly (5 cycles were performed), while transition temperature and the (17) Schild, H. G.; Tirrell, D. A. Macromolecules 1992, 25, 4553–4558.
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size characteristics remained unchanged. Finally, it was found that in solutions of PNIPAM of higher concentration (0.3%) and in the presence of 43 mM of NaCl electrolyte as in the experiment in Figure 2(**), there was no significant effect on the temperature of thermo-induced DNA conformational transition (ΔT < 1 °C), because the concentration of salts much lower than 1 M concentration do not affect LCST of PNIPAM.18 The average size of a DNA globule in fluorescence images before and after heating-cooling is essentially the same; however, due to the limitations of the fluorescence microscopy resolution and the blurring effect, it is not possible to determine the change in morphology of compact DNA after the heating-cooling cycle. To compare the globule morphology of DNA that had been initially compacted by PNIPAM and regenerated after heatingcooling, the samples were analyzed by transmission electron microscopy (TEM). Figure 4A shows a typical electron microscopy image of compacted DNA in a solution of PNIPAM before heating stained by 1% ammonium molybdate. Two types of structures can be clearly distinguished: pale shapeless structures of several tens of nanometers (white arrows) and much larger and intense species (black arrows). The former are considered to be free PNIPAM polymer, while the larger particles are compact DNA condensates, since they have a characteristic size that is typical for compact DNA particles (on the order of 100 nm). In a solution that only contains PNIPAM, only pale structures of several tens of nanometers similar to those indicated by the white arrows in Figure 4A were observed. Next, we observed by TEM the same sample after heating to 37 °C and cooling in the same manner as in the fluorescence microscopy experiment. After chilling, the two similar types of structures were observed by TEM as was shown in Figure 4A. The structures that are attributed to compacted DNA condensates before and after the heating-chilling cycle are compared in Figures 4B and D. In both cases, the compacted DNA morphology is a quasi-spherical particle with a diameter of about 200 nm. Notably, the surface of both DNA globules is also characterized by the pattern shown in Figures 4C and E, which was also observed on the surface of large particles, but not on small ones. The constancy of the shape and size of the compacted DNA morphology indicates that, during the decompaction of DNA and re-entrant collapse by PNIPAM with a decrease in temperature, the structure of compacted DNA remains intact. The proposed mechanism of DNA compaction in PNIPAM and further thermal decompaction is shown in Figure 5. (18) Prevot, M.; Dejugnat, C.; Mohwald, H.; Sukhorukov, G. B. ChemPhysChem 2006, 7(12), 2497–2502.
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Figure 5. Suggested mechanism of DNA compaction and temperature-induced decompaction in solutions of PNIPAM.
Mechanism of DNA compaction is a cooperative influence of the depletion effect in a crowding environment of PNIPAM and a weak interaction between DNA and PNIPAM. An increase in temperature induces a decrease in solubility and the collapse of PNIPAM molecules, which subsequently results in a decrease in polymer crowding as well as decreased availability for interaction of PNIPAM with DNA. Therefore, the compaction of PNIPAM upon heating into globules induces the decompaction of compacted DNA (Figure 5).
activity by external stimuli. Recently, it has been demonstrated that the coil-globule conformational transition of DNA strongly correlates with DNA biological activity, such as transcriptional activity.2,4 Therefore, the “thermal switch” method reported here may be useful for controlling the conformation of genomic DNA to switch DNA genetic activity in an on/off manner. Experimental work in this direction is currently underway.
Conclusion
Acknowledgment. This work was supported by Grant-in-Aid for Scientific Research in Priority Area No. 20034025 (Z.A.) and No. 17076007 (K.Y.) from MEXT, Japan.
In summary, we have shown that the conformation of long genomic DNA in a solution of PNIPAM can be controlled by changing the temperature of the PNIPAM solution. The coilglobule transition of DNA is mediated by the coil-transition of PNIPAM at ca. 33 °C, which is reversible. Our method may be useful in the gene delivery of DNA under conditions in which temperature can be controlled, as well as to control DNA genetic
Supporting Information Available: Supplementary figures of DNA long-axis length distributions in the processes of DNA compaction in solutions of PNIPAM and temperature-induced changes of DNA long-axis length distributions in the solutions of PNIPAM. This material is available free of charge via the Internet at http://pubs.acs.org.
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