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中國機械工程學刊第三十卷第四期第289~295頁(民國九十八年) Journal of the Chinese Society of Mechanical Engineers, Vol.30, No.4, pp.289~295 (2009) Stretching DNA Fibers out of a Chromosome in Solution Using Electro-osmotic Flow Min-Sheng Hung*, Osamu Kurosawa**, Hiroyuki Kabata*** and Masao Washizu**** Keywords : electro-osmosis, DNA, chromosome, laser-induced heating. ABSTRACT This research develops a bio-nanotechnology for mechanically handling individual DNA fibers. The current study particularly focuses on surgery of a chromosome, which is a long strand of DNA tightly wound on proteins. Since DNA wound on a chromosome is several centimeter long, it is too long to observe under a microscope. Therefore, it is necessary to develop a method for partial unwinding. This work uses laser-induced local heating. Focusing a laser beam onto an aimed location on a chromosome immersed in protease solution, induces temperature to rise and locally activates the enzymatic reaction. This work demonstrates stretching DNA fibers out of a chromosome using electro-osmotic flow. Results show a chromosome immobilized onto a glass surface with released DNA fibers as long as 150μm. INTRODUCTION Chromosomal DNA encodes genome information of all the inheritable characteristics of an organic structure. In the stage of post genome handling, DNA information of individual person is suitable to be used for protection or treating diseases. To meet this requirement, the development of bio-nanotechnology allows one to consider conducting bioanalytical assays at very small volumes to increase the speed of these assays and to reduce the amount of material and reagents needed. Paper Received August, 2008. Revised March, 2009, Accepted April, 2009, Author for Correspondence: Min-Sheng Hung. * Assistant Professor, Department of Biomechatronic Engineering, National Chiayi University, Chiayi, Taiwan 60004. ** Researcher, ASTEM RI, Kyoto, Japan. *** Sysmex Co., Kobe, Japan. **** Professor, Department of Bioengineering, The University of Tokyo, Tokyo, Japan. JST, CREST, Japan. Fluorescent in situ hybridization (FISH) is a common technique for locating specific genes on a chromosome (Langer-Safer et al., 1982). The principle of FISH is the hybridization of DNA by using a fluorescence labeled oligonucleotide probes which having the sequences complementary to the unknown DNA sequences. The spatial resolution of FISH depends upon the conformation of the sample DNA. For a closely condensed chromosome, the spatial resolution of FISH may be a few mega base pairs (Alberts, et al., 2002). Heng et al. (1992) demonstrated that the free chromatins released from interphase nuclei for gene mapping. Their results show that the spatial resolution of FISH can approach 10kbp (kilo base pairs) when DNA is extended to a straight fiber. Therefore, physical manipulation of DNA is a useful technique for studying genomic DNA regions. In fact, manipulation of single DNA molecules has been recognized as an important technique in molecular biology. This technique provides a new analytical method completely different from conventional analytical methods in biology and biotechnology. Washizu et al. (1990) proposed and demonstrated a microfluidic technology to stretch and immobilize DNA in the solution by dielectrophoresis. Recently, several studies have focused on manipulation of DNA or physical properties of DNA by using different methods. Wuite et al. (2000) developed and integrated laser trap/flow control video microscope for mechanical manipulation of single biopolymers. For the purpose of understanding their traits, they studied the elasticity of DNA using the combined laminar/optical mode or the flow control system alone to exert the force. Maier et al. (2000) measured the replication rate by a single enzyme of a stretched single strand of DNA. In their study, a strong magnetic field gradient was generated to exert on the magnetic bead and its tethered DNA molecule. Further, the extension of DNA was measured by real-time video analysis of the bead’s image. Bennink et al. (2001) measured force-extension relationships in polymer chains. To be effective, they used optical tweezers setup to attach a streptavidin-covered polystyrene bead to each end of a biotin end-labeled single DNA molecule. -289- J. CSME Vol.30, No.4 (2009) Murayama and Sano (2001) measured the elastic force for a single DNA molecule during a transition between an elongated coil and a collapsed globule state by using dual-trap optical tweezers. Hirano et al. (2002) developed a manipulation technique for native DNA molecules based on laser clustering. They held and manipulated a single DNA molecule with surprising dexterity using a bead cluster created by laser trap. All of the above-mentioned studies were designed to manipulate flexible polymer chains by using a single DNA molecule or single chromatin fibers as their samples. One of the prior studies of stretching DNA as opposed to the conventional approach was reported by Washizu et al. (2003). They presented the application of electro-osmotic flow for stretching out of DNA fibers from a cell. In their study, a yeast cell wall was removed using enzymatic reaction and the cell membrane was dissolved using sodium dodecyl sulfate (SDS) solution wash. The cell prepared was immobilized on a microscope slide having a pair of platinum electrodes. Their results indicated that a yeast cell was immobilized onto a glass surface and the released DNA fibers of as long as 200μm were stretched. Hung et al. (2004) demonstrated that the laser was focused to heat a chromosome in solution. In their study, chromosomal DNA was unwinding by laser-induced heating when the protein degraded enzyme were in solution. Applying force for extending DNA must not exceed DNA fiber mechanical strength (about 100-300pN, Bustamante et al., 2000). Prior studies discuss several methods for extending DNA, including glass needles, magnetic beads, and optical traps. But the above methods cannot sufficiently control force and the DNA extended technique is complex. The aim of the proposed study is to stretch the chromosome to uncurved DNA fibers. In this study, stretching DNA fibers out of a chromosome is demonstrated under a fluorescence microscopy by using electro-osmotic flow. The protocol in the present experiment starts with the locally unwinding of chromosomes, and then stretching chromosomal DNA fibers out of chromosomes. Cathode Debye length Figure 1 Schematic illustration of electro-osmotic flow. the bulk liquid are attracted to the wall and shield these wall charges. Likewise, dissolved co-ions are repelled from the wall. The charged high capacitance region of ions at the liquid/solid interface is called the electric double layer. The ions in the inner layer of counter ions adjacent to the wall are notably immobile. The outer diffuse part of the layer forms a net positive region of ions that span a distance on the order of the Debye length (λd) of the solution. The Debye length λd is defined as λd = 1 2 (e /(εkT))∑ n i zi2 , (1) i where e is the elementary charge, ε is the permittivity of the liquid, k is the Boltzmann constant, T is the temperature, ni and zi are the concentration and the valence of ith ion respectively, and the summation is to be taken over all ionic species present in the liquid. The Deybe length is about 10nm from the wall for symmetric univalent electrolytes at 1mM concentration (Hunter, 1981). Applying an external electric field parallel to the wall causes ions to move in response to the field and drag the surrounding liquid along the wall, called electro-osmotic flow. The velocity profile is governed by STRETCHING DNA BY ELECTRO-OSMOSIS μ The experiment in this work adopts the electro-osmosis method for stretching DNA fibers from a chromosome. This study regards electro-osmosis as a liquid streaming induced by ion drag in the electrical double layer at a solid/liquid interface. Figure 1, shows this charge generation is caused by electrochemical reactions both at the liquid/solid interface and in solid surfaces. The main reaction is deprotonation of acidic silanol groups that produces a negatively charged wall. Counter ions from External field E Anode d 2u dx 2 = ρEE , (2) where the coordinate x is taken perpendicular to the solid surface, E is the field strength, ρE is the charge density, μ is the viscosity of the liquid, u is the liquid velocity parallel to the surface, with the boundary condition u = 0 at the surface and u = u∞ = constant at infinity. Fig. 1 schematically shows the velocity profile caused by electro-osmosis in a solid surface. Because ρE exists only in nm-thickness Debye length above the wall, it creates a very large velocity shear with a constant hydrodynamic drag, regardless of the vertical position of the sample. Changing the -290- M.S. Hung et al.: Stretching DNA Fibers out of a Chromosome in Solution. magnitude and polarity of E also controls the magnitude and direction of the shear. In the flow field, when one end of a chromosomal DNA has a length larger than λd and is immobilized on the solid surface, the DNA fibers extend along the flow with the degree of extension controllable by the applied field magnitude, should a large shear force be applied. USING LASER-INDUCED TEMPERATURE RISE TO PARTIALLY UNWIND A CHROMOSOME The total length of DNA wound on a chromosome ranges from several hundred micrometer of bacterial or eucaryotic cells to several meter of higher species. Since DNA is only 2-nm thick, it is fragile and easily broken even by a gentle flow of the surrounding medium during handling. The present study attempts to extend the whole chromosomal DNA, requiring a method for partially unwinding a chromosome and stretching DNA fibers out of the chromosome. The chromosome is one of the small, rod-shaped, deeply staining bodies that become visible in the eucaryotic cell nucleus at mitosis. Most interphase chromosomes are too far extended and entangled for clearly observing their structures. In contrast, chromosomes from nearly all eucaryotic cells are readily visible during mitosis when they coil up to form highly condensed structures. The chromosome is an intricately folded nucleoprotein complex with many domains, in which the local chromatin structure is devoted to maintaining genes in an active or silenced configuration to accommodate DNA replication, etc. Chromatin is a complex of DNA and proteins in which the genetic material is packaged inside the cells of organisms with nuclei. Thus, the fundamental subunit of chromatin is the nucleosome, consisting of approximately 165 base pairs (bp) of DNA wrapped into two superhelical turns around an octamer of core histones (two each of histones H2A, H2B, H3, and H4), as shown in Fig. 2. Each nucleosome is connected to its neighbors by a short segment of linker DNA and this polynucleosome string is folded into a compact fiber with a diameter of 30nm. The 30-nm fiber is stabilized by a fifth histone, H1, binding to each nucleosome and to its adjacent linker (Alberts, et al., 2002). The molecular surgery of chromosomes requires combining chemical and physical processing. In order to destroy the DNA-protein complex, enzymatic reactions can be used to proceed with this operation; however, the timing and extent of the reaction must be controlled in an appropriate manner. The current investigation uses a laser-induced local Histone DNA Figure 2 Schematic illustration of chromosome and chromatin fiber. Each cylinder represents one histone octamer protein core. The DNA is wrapped around each octamer in a left-handed, superhelical fashion. Chromosome IR Laser Local relaxation of chromosome Pt Electrode Extended DNA Figure 3 Use of laser-induced heating for partially unwinding chromosome and electro-osmotic flow for the extending of a chromosome. heating method for this purpose. Laser is often applied to micromachining using the process of laser ablation (Lai and Huang, 2007; Chou et al., 2008). In the present work, focusing an IR laser beam onto an aimed location on a chromosome immersed in protease solution, induces temperature to rise and activates the enzymatic reaction locally. Figure 3 depicts the chromosome process this study used throughout the experiment. Figure 4 schematically represents the setup used for laser-induced heating, consisting of a fluorescence microscope (OLYMPUS BX-60, Japan) and optical heating with near-infrared light from a CW Raman Fiber laser (1455nm, IPG Laser GmbH, USA). The sample chamber was made of a coverslip and a microscope slide, glued together at four corners of the coverslip with manicure and mounted on a stage which could be moved manually. To visualize the chromosome, the sample was stained by DNA stai- -291- J. CSME Vol.30, No.4 (2009) SIT/CCD RM IP VCR BE DM2 Infrared laser EM EX Hg lamp DM1 Objective Slide / Cover glass Figure 4 Schematic diagram of optical system of laser-induced heating. The infrared laser beam is expanded by beam expander (BE) for adjusting the laser focus to an observation plane of the objective. DM1 and DM2, dichroic mirrors; RM, reflective mirror; IP, image processor. EX is barrier filter to let through the excitation light only and EM the emission light only. ning fluorescence probes YOPRO-1 and 4'-6-Diamidino-2-phenylindole (DAPI) (Molecular Probes Inc.). The transmitted light was collected with an objective (OLYMPUS, IR 100×, oil immersion, NA 1.35, Japan) and imaged onto a SIT (Silicon Intensify Target) camera (Hamamatsu C2400-08, Japan) and CCD camera (Hamamatsu 5810, Japan). The laser-beam path consisted of a 3.3× beam expander and a dichroic mirror. The laser beam focused on the sample using the objective, and the diameter of the laser beam focus was approximately 2μm. The laser light was sent through the beam expander and the objective, and a laser power meter measured the transmitted light intensity. Samples were made consisting of a chromosome protease solution in 20mM MgCl2 solution. The protease solution, used to degrade histones of chromatin, was eukaryal prolyl endopeptidase (PEPase, Pfu Proteinase S, TAKARA Co., LTD.). The optimum temperature for PEPase activity was between 85oC and 90oC with less than 5% of maximal activity observed at room temperature and less than 30% observed at 105oC (Harwood et al., 1997). The process begins with enzymatic local removal of histones from the chromosome with laser-induced heating. The experiment demonstrates partial unwinding of the chromosome by laser-induced heating in PEPase solution. The chromosome used is from human histiocytic lymphoma cells (U-937). The cells are cultured in a 4ml medium (90% RPMI 1640 medium with 10% fetal bovine serum) at 36.2oC. The cell culture is treated and incubated 6 hours with 0.2μg/ml colcemid solution to arrest sufficient cells in metaphase, and harvested for the experiments. The cells are subjected to hypotonic swelling, treated with a detergent, and mechanically disrupted by passage through a fine needle, and the chromosomes are prepared in 20mM MgCl2 solution for the experiments. Figure 5 shows partial unwinding of the chromosome before and after laser-induced heating. The target chromosome is stained by DAPI. The chromosome image is observed using the fluorescence microscope at excitation wavelength 358nm and the fluorescence emission spectra is approximately 461nm. When laser power is 22mW, this study observes local unwinding of the chromosome after laser-induced heating for 3 minutes, as shown in Fig. 5(b). Then laser-induced heating is further increased to 5 minutes, and the chromosomal DNA fibers are cut by UV excitation light, as shown in Fig. 5(c). The excitation wavelength for DAPI is approximately 358nm. The short-wavelength light has stronger energy intensity than the longer-wavelength light. The Fig. 5 result shows that it is easy to destroy the structure for unwinding chromosomal DNA using the short-wavelength excitation for DAPI. Therefore, stai(a) 10µm (b) (c) DNA Figure 5 Unwinding of a chromosome (stained by the fluorescence probe DAPI). The position of the laser spot is indicated by close arrowhead. The photo is taken by CCD camera. -292- M.S. Hung et al.: Stretching DNA Fibers out of a Chromosome in Solution. E (a) stretched DNA (a) (b) shrinking DNA unwinding 10μm 10 μm (b) Photo Figure 6 Partially unwinding of a chromosome (stained by the fluorescence probe YOPRO-1). The position of the laser spot is indicated by close arrowhead. The photo is taken by SIT camera. ning DNA using DAPI is not suitable for stretching DNA fibers from the partially unwinding chromosome. Figure 6 also shows the partial unwinding chromosome before and after laser-induced heating. The target chromosome is stained by YOPRO-1 (excitation 491nm / emission 509nm). The position of the laser beam focus is the point of the arrow indicated in Fig. 6(a). When laser power is 22mW, this study more clearly observes local unwinding of the chromosome after a laser-induced heating for 5 minutes, as shown in Fig. 6(b). The laser beam focused on the chromosome in solution may attribute to temperature increase due to optimum temperature for PEPase activity by light absorption during focus and subsequent heat dissipation to the bulk solution. Fig. 5 and Fig. 6 results prove that laser-induced heating achieves partial unwinding of the chromosome to activate the enzymatic reaction locally. Figure 7 Initial stretching of chromosomal DNA. of the electro-osmotic flow is from anode to cathode. Figure 7 is the photo of the initial stretching process on the left, and its schematic on the right. The photo was taken under a fluorescence microscope. The chromosome is on the left of the photo, and chromosomal DNA fibers are coming out. Carried by electro-osmotic flow, the DNA fibers move to the right in the photo, as shown in Fig. 7(a). When the field is removed, the flexible elastic property of DNA fibers pulls the fibers back near the chromosome, as shown in Fig. 7(b). Results show that electro-osmosis successfully stretches DNA fibers and the results are the same as Washizu et al. (2003) which stretched DNA fibers from a cell. Figure 8 is a photo of a larger area, the photo on the top and its schematic on the bot- Stretched DNA STRETCHING CHROMOSOMAL DNA chromosome Stretching of chromosomal DNA by the electro-osmotic flow is experimentally demonstrated. For human cells, the physical length of homologous chromosomes is approximately 4-5cm. The chromosome prepared is immobilized on a microscope slide having a pair of platinum electrodes. A mild treatment with PEPase and laser-induced heating is done to partially unwinding the chromosome, and the field of about 1kV/m is applied. When the voltage is applied, electro-osmotic flow is induced, and the chromosomal DNA is pulled out of the chromosome. It should be noted that the glass surface in water is positively charged, so the direction Schematic 10μm chromosome Figure 8 Stretching of chromosomal DNA up to 150μm. Top: photo, Bottom: schematic. -293- J. CSME Vol.30, No.4 (2009) tom. Observations show DNA fibers coming out from the chromosome, which is stretched as long as 150μm. The extended length is much shorter than the maximum length DNA in the chromosome. This result may be caused by incomplete stretching due to the chromosome not unwinding enough, or extended DNA fibers cut by the excited fluorescence. CONCLUSIONS The present study investigates the extending of whole chromosomal DNA from a singular molecular chromosome. However, human homologous chromosomes are complex and consist of both DNA and proteins. Therefore, enzymatic digestion and laser-induced heating are used to take apart the DNA-protein to partially unwind chromosomes due to its complexity. The stretching of DNA is made with the use of electro-osmotic flow, which generates a high velocity shear near the surface of the glass where chromosomes are attached. In the present study, the stretching of the chromosome is experimentally demonstrated by using human homologous chromosomes as sample. Our future work will improve DNA fiber’s positioning and anchoring for chromosomal DNA to extend enough in solution to investigate FISH resolution. ACKNOWLEDGEMENTS This work was supported in part by BRAIN (Seiken Kiko) Research and Development Program for New Bio-industry Initiatives, and the National Science Council (NSC 93-2313-B-415-003). REFERENCES Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P., Molecular Biology of the Cell, 4th ed, Garland Science, New York, pp. 500, 208-231 (2002). Bennink, M. L., Leuba, S. H., Leno, G. H., Zlatanova, J., Grooth, B. G. De, and Greve, J., “Unfolding Individual Nucleosomes by Stretching Single Chromatin Fibers with Optical Tweezers,” Nature Structural Biology, Vol. 8, pp. 606-610 (2001). Bustamante, C., Smith, S. B., Liphardt, J., and Smith, D., “Single-Molecule Studies of DNA Mechanics,” Current Opinion in Structural Biology, Vol. 10, pp. 279-285, (2000). Chou, X.-R., Wang, J.-X., and Chen, J.-S., “Laser Direct Writing of Diamond Micro-nozzle Using Q-Switched Nd:YAG Laser,” Journal of the Chinese Society of Mechanical Engineers, Vol. 29, pp. 289-297, (2008). Harwood, V. J., Denson, J. D., Robinson-Bidle, K. A., and Schreier, H. J., “Overexpression and -294- Characterization of a Prolyl Endopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus,” Journal of Bacteriology, Vol. 179, pp. 3613-3618 (1997). Heng, H. H.Q., Squire, J., and Tsui, L.-C., “High-Resolution Mapping of Mammalian Genes by in situ Hybridization to Free Chromatin,” Proceeding of the National Academy of Sciences, Vol. 89, pp. 9509-9513 (1992). Hirano, K., Baba, Y., Matsuzawa, Y., and Mizuno, A., “Manipulation of Single Coiled DNA Molecules by Laser Clustering of Microparticles,” Applied Physics Letters, Vol. 80, pp. 515-517 (2002). Hung, M.-S., Kurosawa, O., Kabata, H., and Washizu, M., “An Integrated Laser-Induced Heating/Electroosmosis System for the Molecular Surgery of Chromosomes”, Proc. of CSME the 21th National Conference on Mechanical Engineering, Kaohsiung, Taiwan, (2004). Hunter, R. J., Zeta Potential in Colloid Science: Principles and Applications, Academic Press, New York (1981). Maier, B., Bensimon, D., and Croquette, V., “Replication by a Single DNA Polymerase of a Stretched Single-Stranded DNA,” Proceeding of the National Academy of Sciences, Vol. 97, pp. 12002-12007 (2000). Murayama, Y., and Sano, M., “Force Measurements of a Single DNA Molecule,” Journal of the Physical Society of Japan, Vol. 70, pp. 345-348 (2001). Lai, H.-Y. and Huang, P.-H., “Molecular Dynamics Analyses of the Femtosecond Laser-induced Grain Boundary Spallation,” Journal of the Chinese Society of Mechanical Engineers, Vol. 28, pp. 577-583, (2007). Langer-Safer, P. R., Levine, M., and Ward, D. C., “Immunological Method for Mapping Genes on Drosophila Polytene Chromosomes,” Proceeding of the National Academy of Sciences, Vol. 79, pp. 4381-4385 (1982). Washizu M. and Kurosawa, O., “Electrostatic Manipulation of DNA in Microfabricated Structures,” IEEE Transactions on Industry Applications, Vol. 26, pp. 1165-1172 (1990). Washizu, M., Nikaido, Y., Kurosawa, O., and Kabata, H., “Stretching Yeast Chromosomes Using Electroosmotic Flow,” Journal of Electrostatics, Vol. 57, pp. 395-405 (2003). Wuite, G. J. L., Davenport, R. H., Rappaport, A., and Bustamante, C., “An Integrated Laser Trap/Flow Control Video Microscope for the Study of Single Biomolecules,” Biophysical Journal, Vol. 79, pp. 1155-1167 (2000). NOMENCLATURE M.S. Hung et al.: Stretching DNA Fibers out of a Chromosome in Solution. E e k n u T x electric field, [V/m] elementary charge, [C] Boltzmann constant, [J/K] concentration of ion, [mol/m3] liquid velocity parallel to the surface temperature, [K] Cartesian coordinate is taken perpendicular to the solid surface z valence of ion Greek Symbols ε permittivity of medium, [C/Vm] electric charge density, [C/m3] ρE μ viscosity of the liquid, [pas] λd Debye length, [m] Σ summation of over all ionic species Subscripts i with reference to ith species 以電滲流拉伸溶液中之染 色體 DNA 洪敏勝 國立嘉義大學生物機電工程學系 黑澤修 日本(財)京都高度技術研究所 加畑博幸 日本 Sysmex 股份有限公司中央研究所 鷲津正夫 日本東京大學生物工程學系 日本科學技術振興機構, CREST 摘 要 本研究為建立一套利用物理方法操作 DNA 之 生物奈米技術,主要實驗對象為真核細胞染色體。 由於真核細胞染色體是由 DNA 與組蛋白纏繞而形 成,必須將染色體上某一部位之 DNA 鬆弛,才能 進行 DNA 之操作。故本研究使用於物鏡下聚焦之 雷射間接加熱方式,在含有高溫時具較大活性之組 蛋白分解酵素之溶液中直接加熱染色體,並以電滲 流拉伸染色體 DNA。研究結果顯示染色體可被固 定於玻璃基板上,且電滲流可順利將 DNA 拉伸, 其拉伸之長度約可達到 150µm。 -295-