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Giant Liposome as a Biochemical Reactor: Transcription of DNA and Transportation by Laser Tweezers Kanta Tsumoto,†,‡ Shin-ichirou M. Nomura,‡ Yoichi Nakatani,§ and Kenichi Yoshikawa*,‡ Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan, Department of Physics, Graduate School of Science, Kyoto University, and CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation, Sakyo, Kyoto 606-8502, Japan, and Laboratoire de Chimie Organique des Substances Naturelles associe´ au CNRS, Centre de Neurochimie, Universite´ Louis Pasteur, 5 rue Blaise Pascal, 67084 Strasbourg, France Received June 12, 2001. In Final Form: July 26, 2001 Transcription of T7 DNA has been performed within cell-sized (∼10 µm) liposomes that were formed by natural hydration of phospholipid films. From direct observation by fluorescence microscopy, it was confirmed that the cell-sized liposome behaves as a barrier that can prevent the attack of RNase in the bathing solution surrounding it. It is shown that the liposome encapsulating the transcripts can be transported by use of laser tweezers. Such newly developed procedures on cell-sized liposomes would have applications in the microlaboratory of biochemistry and molecular biology.
Introduction Currently, in vitro experiments of chemistry and biochemistry have mostly been carried out within “hard” vessels, or glass/plastic apparatus, sized 1-100 mm. However, in life almost all essential reactions proceed within “soft” compartments, or closed biomembranes with sizes of 0.1-10 µm. During the past decade, there has been increasing interest in downsizing the scale of laboratory experiments, such as combinatorial chemistry, micro-sized chips fabricated for DNA analysis, and so forth. Application of lithography techniques to the fabrication of micrometer-sized experimental systems seems useful to realize “microexperimental laboratories” on a solid plate. Actually, much effort has been paid to elaborate micropump, microreactor, microdetector, and flow networks with channel widths and depths on the micrometer scale.1 Although such attempts seem important, it is becoming clear that the cost and the necessary efforts increase steeply with the decrease of the system size. It does not seem easy to construct and arrange the individual elements of a microlaboratory down to the scale of micrometers. In addition, the effect of the surface of a “solid vessel” becomes increasingly significant as the vessel size decreases. The ratio of surface area to volume, A/V, increases in inverse proportion as the size of “vessels” decreases. Any kind of solid surface leads to interference effects with chemicals, especially with macromolecules. Surface adsorption and denaturation are phenomena frequently encountered on solid surfaces. This means that micrometer-sized reactors and flow channels fabricated on a substrate exhibit serious problems due to surface effects. With this in mind, it would be of value to examine the feasibility of cell-sized liposomes as small “test tubes” * To whom all correspondence should be addressed. E-mail:
[email protected]. Phone: +81-75-753-3812. Fax: +81-75-753-3779. † Nagoya University. ‡ Kyoto University and CREST of Japan Science and Technology Corp. § Universite ´ Louis Pasteur. (1) Bruin, G. J. M. Electrophoresis 2000, 21, 3931-3951.
in a microlaboratory, because life utilizes micrometersized bags of lipid membranes, avoiding harmful effects from surfaces. Usually in most previous studies, small liposomes (submicrometers) were used as microreactors.2 However, there have been some pioneering studies utilizing giant liposomes of phospholipids as a model for living cells. For example, morphological change in a cell-sized liposome has been observed accompanied by the polymerization reaction of actin or tubulin inside the liposome.3 Injection of chemical compounds into liposomes has also been studied by use of micrometer-sized glass pipets.4 Although these studies have demonstrated good visual examples of the utility of liposomes, there still remain various technical problems in order to realize microscaled laboratories by application of cell-sized liposomes. For example, we have to find the experimental methodology: (i) how to obtain stable cell-sized liposomes, (ii) how to proceed with chemical/biochemical reactions within the liposomes, and (iii) how to transport the desired liposome to the desired position on a microlaboratory. In the present paper, we report our experiments on giant liposomes, showing (i) stable cell-sized liposomes containing the necessary species for RNA polymerization, (ii) direct observation of RNA molecules synthesized inside the liposome, and (iii) transportation of the liposome encapsulating reaction products by using laser tweezers. Experimental Section Transcriptional Reaction inside a Giant Liposome. Coliphage T7 DNA (∼38 kbp), purchased from Sigma Chemical Co., and T7 RNA polymerase, purchased from Life Technologies, were used without further purification. A solution of T7 DNA (0.9 ng/µL) as template and T7 RNA polymerase (0.5 U/µL), containing 40 mM Tris-HCl, 0.1 M sucrose, 8 mM MgCl2, 2 mM spermidine‚3HCl, 25 mM NaCl, 5 mM dithiothreitol (DTT), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, and 10 µM UTP (all (2) Walde, P. Curr. Opin. Colloid Interface Sci. 1996, 1, 638-644. (3) Hotani, H.; Nomura, F.; Suzuki, Y. Curr. Opin. Colloid Interface Sci. 1999, 4, 358-368. (4) Wick, R.; Angelova, M. I.; Walde, P.; Luisi, P. L. Chem. Biol. 1996, 3, 105-111.
10.1021/la010887s CCC: $20.00 © 2001 American Chemical Society Published on Web 09/07/2001
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Figure 1. Fluorescence images with the time interval of 0.85 s on a giant liposome entrapping T7 DNA and the transcripts. (A) Fluorescence images of DAPI on T7 DNA entrapped in a giant liposome. (B) Fluorescence images of BODIPY on RNA in the same liposome as (A). The images were processed with pseudo color. The rightmost picture represents the profile of fluorescence intensity of the liposome at 0 s. ribonucleotides were purchased from Roche Diagnostics, as lithium salt), was used for the transcriptional reaction. To visualize the RNA and DNA, we used 1 µM fluorochrome-labeled UTP (ChromaTide BODIPY TMR-14-UTP (BODYPI-UTP), Molecular Probes) and 0.6 µM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, fluorochrome for staining DNA), respectively. A similar method for visualization of the ongoing transcription on a single DNA chain was first adopted by the group of Place et al.5 We have recently established the methodology to prepare cellsized liposomes from neutral phospholipids through natural swelling of dry lipid films.6-8 In the encapsulation of the transcriptional reaction, however, we have encountered a difficulty in preparing stable cell-sized liposomes from neutral phospholipids under the procedure followed in the previous studies.6-8 Thus, we have explored the experimental conditions to obtain the giant liposome for the observation of the transcriptional reaction inside it. Consequently, an adequate yield of liposomes could be obtained by employing lipid films containing negatively charged phospholipid. That is, an aliquot (25 µL) of 1 mM dioleoyl-phosphatidylcholine (DOPC, Sigma) and 0.1 mM dioleoyl-phosphatidylglycerol (DOPG, Sigma) in 1:2 (v/v) methanol/chloroform in a glass test tube was allowed to stand still overnight under vacuum-drying. The lamellar solid film thus obtained was swelled with 25 µL of the solution for the transcriptional reaction for up to 24 h at 4 °C. The swelling was performed at low temperature in order to inhibit transcription during the swelling procedure of the lipid film. For the digestion of transcripts by ribonuclease, an aliquot of ribonuclease (2 µg/ mL; RNase from Bovine Pancreas, DNase free; NIPPON GENE) in the transcriptional reaction solution without RNA polymerase was placed in contact with the solution containing the giant liposomes on the stage of the microscope. Fluorescence Microscopic Observation. We used an inverted fluorescence microscope (Carl Zeiss Axiovert 135 TV) equipped with a high-sensitivity Hamamatsu Photonics SIT TV camera. The fluorescence images of T7 DNA and nascent RNAs were obtained by excitation of DAPI and BODIPY, respectively, and recorded on an S-VHS videotape at 30 frames/s. Lipid (5) Place, C. Ecole Normale Supe´rieure de Lyon. Personal communication. (6) Magome, N.; Takemura, T.; Yoshikawa, K. Chem. Lett. 1997, 205-206. (7) Kumazawa, N.; Mel’nikova, Yu. S.; Yoshikawa, K. Macromol. Symp. 1996, 106, 219-223. (8) Nomura, S.-i.; Yoshikawa, K. In Giant Vesicles, Perspectives in Supramolecular Chemistry; Luisi, P. L., Walde, P., Eds.; John Wiley & Sons, Ltd.: Chichester, 2000; Vol. 6, pp 313-317.
Figure 2. Protection of RNA encapsulated in a giant vesicle against digestion by RNase. The images labeled “giant liposome” indicate giant liposomes encapsulating T7 DNA and RNA transcripts. The images labeled “glass surface” indicate T7 DNA and RNA transcripts adsorbed on the glass. membranes were also stained simultaneously with these fluorochromes. The apparent conformation and spatial position of individual DNA molecules and the fluorescence signal of nascent RNA molecules on the video frames were calibrated with an image
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Figure 3. Transportation of a giant liposome encapsulating transcripts by use of laser tweezers. (A) Serial fluorescence images of BODIPY on a giant liposome. (B) Transportation of the giant liposome encapsulating the transcripts as observed with DAPI fluorescence. The open arrow indicates the visible DNA molecule. (C) Serial fluorescence images of the giant liposome after the transportation. (D) Schematic representation of the transportation of a liposome with laser tweezers. A giant liposome was trapped on the fixed focus of the laser introduced through the objective lens. Here, transportation was performed by moving the microscope stage laterally, from A to C through B.
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processor (Argus 20, Hamamatsu Photonics). An image processor (Cosmos; LIBRARY, Tokyo) was used to obtain better contrast. For laser trapping, a 1064 nm laser beam (CW Nd3+:YAG laser, 250 mW, Spectron, SL-902T) was focused on the stage of an inverted laser microscope (Nikon, TE-300).
Results and Discussion Figure 1 shows the fluorescence microscopic images of a single T7 DNA (Figure 1A, DAPI, upper row) and RNA transcript (Figure 1B, BODIPY, lower row) in a giant liposome prepared through natural swelling of lipid films with the reaction solution. Hydration of lipid films and swelling of liposomes proceeded overnight at 4 °C, the low temperature inhibiting transcription.9 We then transferred prepared giant liposomes encapsulating the reaction solution onto the stage of the microscope at room temperature (ca. 22 °C). Thus, giant liposomes were expected to form with the necessary materials inside themselves before transcription started. We have confirmed that giant liposomes encapsulating the reaction solution remain stable at 4 °C for at least 1 week after formation (data not shown). The corresponding quasi-three-dimensional representation of the fluorescence intensity profile about the leftmost picture in each row indicates the existence of an entrapped single DNA molecule. The series of images show that the entrapped T7 DNA together with the transcripts exhibits Brownian motion within the liposome by avoiding adsorption onto the inner membrane surface. Here, RNA transcripts are observed in the bound state to T7 DNA. The transcripts released from RNA polymerase are not visible in the figure, due to the small size of free RNA molecules compared to those connected to the template DNA. It is to be noted that liposomes encapsulating DNA together with RNA transcripts are obtained in very mild conditions, that is, natural swelling, without any harmful treatment such as utilization of an organic solvent, surfactant, or freeze-thawing procedure.10 Figure 2 shows the fluorescence images of a giant liposome encapsulating the DNA and transcripts, together with the observation on the glass surface in the same bathing solution, with and without the addition of RNase. RNA molecules adsorbed on the glass surface were completely digested with the addition of RNase. In contrast, the RNA transcripts inside the liposome remained safe even after the addition of RNase, suggesting that the liposome membrane can play the role of an impermeable wall to prevent the entry of RNase into the liposome. Figure 3 exemplifies the transportation of a giant liposome encapsulating the transcriptional reaction by use of laser tweezers. The focused laser can trap the lipid membrane, because of its higher permittivity compared to the solvent (H2O).11 Figure 3A shows the time series of the fluorescence microscopic images of nascent RNA transcripts in a liposome, exhibiting Brownian motion. Figure 3B shows the process of transportation of the giant liposome, where the microscope stage was moved laterally as is schematically represented in Figure 3D. The giant liposome was transported at the rate of 5-50 µm/s. Once (9) Oakley, J. L.; Strothkamp, R. E.; Sarris, A. H.; Coleman, J. E. Biochemistry 1979, 18, 528-537. (10) Barenholz, Y.; Lasic, D. D. In Handbook of Nonmedical Applications of Liposomes; Barenholz, Y., Lasic, D. D., Eds.; CRC Press: Boca Raton, FL, 1996; Vol. 3, pp 23-30. (11) Bar-Ziv, R.; Moses, E.; Nelson, P. Biophys. J. 1998, 75, 294320.
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giant liposomes are trapped by laser, they are usually easy to transport as far as subcentimeters to centimeters. During the transportation of the liposome, the image of the RNA transcripts was hard to observe. Thus, in the figure, the DAPI image on DNA is shown for the visualization of the transportation. Figure 3C shows that the RNA transcripts remain within the liposome even after the transportation with the laser tweezers. In this work, we have shown the preparation of a giant liposome encapsulating a “biochemical reaction”, protecting the reaction products from the enzyme outside. It is also shown that a giant liposome can be transported safely in an aqueous solution. This suggests that a giant liposome can serve as a micrometer-scale test tube in a microlaboratory. To obtain giant liposomes encapsulating the desired solution, the current methodology includes electroformation of giant liposomes12 and microinjection into them.4 Although such a method has been shown to work, it requires a skillful technique. On the other hand, the method of natural hydration, reported here, is easy to perform; through natural swelling of lipid films alone, one can obtain stable giant liposomes entrapping the reaction solution. Using laser tweezers, one can choose a desired giant liposome from a pool of them and transport it individually under observation. It is, of course, useful to directly manipulate liposomes with micromanipulators that can generate a stronger force than laser tweezers,13 but here we adopted them because of their advantages in dealing with encapsulated reactions, namely, no necessity of chemical modification and no invasion into the interior during transportation. In recent papers, we have demonstrated that long DNA molecules can be encapsulated into giant liposomes in a good yield through a natural swelling method.8,14 The present paper shows that the incorporated DNAs can be further transcribed into RNAs in such cell-sized liposomes. By applying the experimental procedures reported here, it is expected that individual liposomes will serve as a transporter and simultaneously as a reactor in a microlaboratory. Considering the fact that the microscale space compartmentalized by phospholipid membranes is one of the essential structures in life, the studies of biochemistry and biophysics inside the small space of a liposome should be promising. Such studies will also contribute fundamental insight into the challenge of how cells originate de novo.15 Acknowledgment. We thank Professor M. Sugiura, Nagoya City University, for his helpful discussion and Professor G. Ourisson, Universite´ Louis Pasteur, for his critical reading of the manuscript. S.-i.M.N. is supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (No. 3257). LA010887S (12) Angelova, M. I.; Sole´au, S.; Me´le´ard, Ph.; Faucon, J. F.; Bothorel, P. Prog. Colloid Polym. Sci. 1992, 89, 127-131. (13) Stro¨mberg, A.; Ryttse´n, F.; Chiu, D. T.; Davidson, M.; Eriksson, P. S.; Wilson, C. F.; Orwar, O.; Zare, R. N. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7-11. (14) Nomura, S.-i. M.; Yoshikawa, Y.; Yoshikawa, K.; Dannenmuller, O.; Chasserot-Golaz, S.; Ourisson, G.; Nakatani, Y. ChemBioChem 2001, 2, 457-459. (15) Szostak, J. W.; Bartel, D. P.; Luisi, P. L. Nature 2001, 409, 387390.
