Applications of electrostatic stretch-and-positioning of DNA - IEEE Xplore

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Applications of Electrostatic Stretch-and-Positioning of DNA Masao Washizu, Member, ZEEE, Osamu Kurosawa, Ichiro &ai,

Nobuo Shimamoto

Seiichi Suzuki, and

Abstmcf-The authors have previously reported that the elecX-phage DNA :48.5 kb (16.2 p) trostatic orientation and the dielectrophoresis (DEP) of DNA 4 * occur under szl MHz, > 1 x 10’ Vlm field, by which DNA strands are stretched straight along field lines and positioned (0.5 pm) 30kb (10.0 pm) Y onto electrode edges. This paper presents some application of this stretch-and-positioning method to genetic engineering. It is shown that the DNA size distribution, as well as the activitiesof nuclease, restriction sites of Kpn I can be determined by the measurement of the apparent length 5.1 pm of stretched DNA. Several methods are developed to immobilize stretched DNA onto a substrate, including: 1) immobilization 0.5 p 10.0 p onto a conducting substrate for observations with the scanning 6.2 pn . I 10.5 pn tunneling microscopy, 2) anchoring onto a substrate only at the both ends of DNA using special electrode configuration, and/or I molecular binding between avidin and biotin. The DNA can be Fig. 1. Locations of restriction sites of bacteriophage X DNA for K p n I. held without contact to the substrate in the latter method, so that it does not cause steric hindrances to the DNA-binding enzymes. A novel Fluid Integrated Circuit (FIC) device is proposed in analysis of higher species. For instance, human genome conwhich stretched DNA is cut by laser beam for the successive sists of 3 x lo9 base-pairs, which would require more than sequencing. A method to obtain unidirectionally oriented DNA lo7 times of subcloning and electrophoretic operations for the is developed. The spatial resolution, and the small number of molecules re- determination of all sequences. Another inherent problem associated with such a chemical quired, are the advantages of the assays and measurements using electrostatic DNA manipulations over conventional biochemical method is that it lacks spatial resolution. A DNA molecule is methods. It is hoped that the methods may open a way to a novel handled as fragments in aqueous solution, and the information category of “molecular biochemistry with spatial resolution.” about the location of a particular sequence in the original DNA strand is usually lost. For instance, consider the case of sequencing large DNA. Because only 0.5 kb can be sequenced I. INTRODUCTION ETERMINATION of a DNA sequence constitutes an at a time using electrophoresis, the DNA is first enzymatically important part of molecular biology. The conventional cut into fragments smaller than 0.5 kb, subcloned, and the sequencing relies on gel electrophoresis: DNA samples are sequence of each fragment is determined. The full sequence elongated till every position (Sanger method), and the sizes of the original DNA is inferred from the sequence of each of the resultant fragments are analyzed by the difference in fragment. The reconstruction is like solving a jigsaw puzzle, the electrophoretic mobility on a gel plate, from which the and becomes increasingly difficult as the size of the DNA insequence is deduced [ 11. This method typically requires DNA creases. If the information about the location of each fragment samples of the order of micrograms, and the sequence of about is preserved, this reconstruction step may be greatly simplified. The authors have been engaged in the development of 0.5 kilo base-pairs (kb) is determined at one time. The method, however, is not rapid enough, in particular for the genetic electrostatic manipulation techniques of DNA using M 1 MHz > 1 x lo6 Vlm field created in microfabricated electrode systems [ 2 ] , and have shown that: Paper MSDAD 93-105, approved by the Electrostatic Processes Committee of the IEEE Industry Applications Society for presentation at the 1993 IEEE 1) A DNA molecule, randomly coiled under the absence Industry Applications Society Annual Meeting, Toronto, Ontario, Canada, of the electrostatic field, is stretched straight along the October 3-8. The work is supported in part by the grants of Advance Company, Toyota Physical and Chemical Research Institute, the Ministry of field lines. Education to M.W., and the Agency of Science and Technology of Japan to 2) Using a vacuum-evaporatedparallel strip electrodes with N.S., and the research project of the National Institute of Genetics. Manuscript sharp edges, the stretched DNA is attracted to where released for publication November 28, 1994. M. Washizu and S . Suzuki are with Seikei University, Tokyo 180 Japan. the field is stronger by dielectrophoresis (DEP, [3], [4]), 0. Kurosawa is with Advance Company, Tokyo 103 Japan. until one end of the molecule touches the electrode I. Arai was with Seikei University, Tokyo 180 Japan. He is now with edge, while the other end extends perpendicular to the the Technological Development Department, Nikon Corporation, Tokyo 140, Japan. electrode. N. Shimamoto is with the DNA Research Center, National Institute of This method, hereafter referred to as “electrostatic stretch-andGenetics, Mishima, Shizuoka 41 1 Japan. IEEE Log Number 9409656. positioning of DNA,” can potentially enable manipulations of

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Fig. 2. Electrostatically stretch-and-positionedrestriction fragments.

DNA in a single molecule level with spatial resolution, which have not been attainable with chemical methods. This paper presents some applications of the method, together with the proposals of novel techniques for gene sequencing and biophysicalhiochemical researches. 11. SIZEDETERMINATION OF DNA

Gel electrophoresis is widely used for the determination of DNA size due to its simplicity and high resolution [5]. However, there are still technical problems in that: 1) the analysis takes hours, and 2) the resolution becomes poorer for DNA larger than 10 kb. The authors have previously proposed a new method of the size-measurement, in which DNA size is determined from the electrostatically stretched length using the structural constant (length per bases) 3.0 [kblpm] [2]. This constant is for right-handed, B-type double-stranded DNA, which is the typical DNA conformation in water [6]. Here, the method is applied for the determination of the size-distribution of DNA. The principle is as follows: First, DNA fragments with an unknown size-distribution are stained with fluorescent dye. Then they are brought into microfabricated parallel-strip electrode gap, and are stretch-and-positionedonto the electrode edge. Because all DNA molecules, independent of their length, are aligned perpendicular to the electrode with one end touching to the edge at this stage, the fluorescent intensity is highest at the electrode edge, and becomes lower apart from the edge where shorter DNA cannot reach. Therefore, by measuring fluorescent intensity as a function of the displacement from the electrode edge, DNA-size distribution can be determined. The above argument is under an assumption that a DNA molecule is uniformly fluorescent-labeled along its length, so a precaution must be paid for the choice of the fluorescent dye. 4', 6-diamidino-2-phenyl-indole(DAPI) used in [2] is not suitable, because it binds preferentially to A-T pairs rather than G-C pairs. We used acrydine orange, which intercalates into the bases, and realizes sequence-independent staining.

The method is demonstrated with the restriction fragments of bacteriophage X DNA [7]. The restriction enzyme, Kpn I, cuts X DNA at two sites, as shown in Fig. 1. If DNA is partially digested, the random digestion will produce fragments of 10.5, 10.0,6.2,5.7, and 0.5 pm,and there will be undigested DNA with the full length of 16.2 pm, as shown in Fig. 1. The photo of the stretched DNA fragments in Fig. 2 in fact shows different sized fragments. (Note: Individual DNA molecules are not seen in the photo: regular vertical lines in this figure, horizontal lines in Figs. 5 , 7, 9, and 14 are video-scanning. The curved lines in Fig. 11 are single DNA molecules). The quantitative measurement of the size distribution is made by measuring the fluorescent intensity along the DNA length using a photomultiplier. The dimension of a rectangular window through which the light signal is fed to the photomultiplier (see Fig. 2), is chosen based on the following considerations: The width of the window parallel to DNA is made small enough for better resolution, and the perpendicular width is taken somewhat larger to accept sufficient fluorescence for the measurement, and also to take average over a certain number of molecules. Three long fragments 6, 10, and 16 pm, are detectable, as seen in Fig. 3. The intensity in the ordinate indicates relative amounts of the fragments. Due to the limitation in the optical resolution, the shortest fragments were not detected. The accuracy of this DNA-size measurement depends on several factors including: 1) the field strength, 2) the conductivity of the medium, and 3) the type of fluorescent dyes used. The stretched length of a DNA, a string-like flexible molecule, is determined by the equilibrium between the electrostatic force and the thermal randomization, and the stretched length asymptotically approaches to the molecular length as the electrostatic stress becomes stronger. Our experiments using DAPI as the fluorescent dye show that the conductivity should be less than 2 pS/cm to obtain conductivity-independent stretched length, and, even in this case, about 2% increase in the stretched length is observed when the field is raised

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from 1 x lo6 V/m to 1.5 x lo6 V/m. It is also observed that DNA strand is elongated by several percent when stained with intercalating dies such as ethidium bromide. Therefore, when higher accuracy is required, DNA with known size, labeled with the same fluorescent dye, should be added as the standard, and the measured size is to be evaluated relative to this standard size. The advantages of the method are that it is fast, and can handle large DNA of several hundred kilo-base pairs. The optical resolution limit of the method may not be a serious problem for long DNA where relative accuracy is still ensured.

Fig. 5. Stretch-and-positionedA DNA after 0, 3. 6, and 8 h of exonuclease treatment.

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DNA : b DNA

111. MEASUREMENT OF NUCLEASE ACTIVITY

An exonuclease digests DNA from its ends. The activity of the enzyme is defined as the number of mononucleotides removed in a unit time, so by measuring the shortening of the DNA length as a function of digestion time, the activity can be determined. In the experiment presented here, digestion of A-phage DNA with T4 DNA polymerase is measured. T4 DNA polymerase has an exonuclease activity when free nucleotides are absent, and digests DNA from 3' end toward 5' end [8] (see Fig. 4). For a double stranded DNA with the initial length Lo [pm], the digestion occurs from both ends, and after t [h] of reaction, there will be single-stranded portions of the length L, = ut at both ends, and a double-stranded portion of the length

L d = Lo - 2ut

(1)

at the center, where U is the digestion speed [pmh]. The double-stranded part can be distinguished from the singlestranded part by the observation under a fluorescent microscope, because binding of most fluorescent dyes to singlestrands is much weaker than that to the double-strands. Fig. 5 shows the photographs of electrostatically stretchand-positioned DNA stained with DAPI after 0, 3, 6, and 8 h of digestion. Fig. 6 plots the length as a function of the

0

2

4

6

8

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Reaction time [hrs] Fig. 6. Measurement of an exonuclease activity by the electrostatic stretchand-position method.

reaction time, measured from the photos in Fig. 5. The slope of the plot is twice the digestion speed, as shown in (1). A question we had addressed was whether or not the single-stranded portion of DNA is subject to the electrostatic orientation and the DEP effects. We are unable to perform a direct observation, because efficient single-strand staining fluorescent probes are unavailable. Acrydine orange and some other dyes are supposed to bind also to single-stranded DNA, but they do not have fluorescent yield high enough to visualize DNA in a single molecular level. The answer is given in Fig. 5. If the single-stranded part is stretched under the electric field and influenced by DEP just as the double-stranded part, then the double-stranded part of the stretched-and-positioned DNA in Fig. 5 should lie apart from the electrode edges with the distance ut [pm]: This distance is where the singlestranded part should be, which is invisible. However, all visible portions (i.e., double-stranded portions) in Fig. 5 are in direct

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contact with the electrode edge. Therefore, we concluded that, under 1 MHz 1 x lo6 V/m electric field, single-stranded DNA is not stretched. This is probably because single-stranded DNA is packed into coiled conformation by the hydrophobic interactions of the bases, or by forming base-pairs within the single strand. On the other hand, double-stranded DNA has hydrophilic backbones and the bases are not exposed to surrounding water, so that it is more likely to take stretched conformations.

IV. ELEC’ROSTATIC IMM~~ILIZATION OF DNA When the electrostatic method is used in combination with biochemical techniques, a problem due to the conductivity of the medium arises. An enzymatic reaction must be performed in a buffer specific to each enzyme, which in most cases is a physiological saline solution. On the other hand, the electrostatic orientation and the dielectrophoresis of DNA occurs only in a medium with resistivity higher than several hundred k 0 cm. This problem is solved by a stepwise procedure, including: 1) electrostatic stretch-and-positioning,2) exchange of the medium, and 3) biochemical reaction. DNA must be immobilized tightly enough, and the medium exchange must be gentle enough to retain DNA against flow. This requirement led us to develop some immobilization techniques of the electrostatically stretch-and-positioned DNA.

Fig. 7. DNA ionically immobilized onto a glass substrate.

(b)

A. Immobilization of DNA on insulating and Conducting Substrate

Fig. 8. FIC device for the immobilization of DNA on a conducting surface.. (a) Top view. (b) Cross-sectional view.

One end of the electrostatically stretch-and-positionedDNA, which is in touch with a vacuum-evaporated aluminum electrode edge, tends to be fixed permanently. The binding is so strong that when a flow of the medium is applied, DNA breaks in the middle rather than dissociate from the contact point. The mechanism is not known, but judging from the strength of the binding, we assume that a covalent bond is formed between the DNA and the metallic electrode surface. Phosphates at 5‘ end of a DNA molecule might be involved in this process. The above phenomenon makes it possible to immobilize DNA molecules at the electrode edge. It is experimentally observed that the adhesion of the molecular end to the electrode edge more easily occurs, and is stronger, with aluminum electrodes than with platinum or gold electrodes. This, and the fact the fabrication of the micro-electrodes is easier with aluminum, led us to use aluminum electrodes for the DNA immobilization. However, by using a simple parallel strip electrode pair, only one end of DNA can be fixed onto the electrode edge. When the field is turned off, the DNA stretched by the electric field shrinks back to random-coil conformation, with an end fixed at the electrode edge and the other making Brownian motion. Some additional techniques are needed to retain the stretched conformation after the field is removed. The simplest way to fix stretch-and-positioned DNA onto a substrate, regardless of how they are fixed, is to use ionic interactions. A DNA molecule has a negatively charged phosphate backbone, and it has a tendency to be adsorbed onto a positively charged surfaces. Glass has a negative surface

charge, so the addition of divalent positive ions, such as Ca++ or Mg++, is effective in fixing DNA onto a glass substrate [9]. The divalent cations act as an adhesive between negatively charged DNA and the negatively charged substrate. The amount of ions added must be at a minimum to keep the low conductivity of the medium. Fig. 7 shows a photograph of thus fixed DNA. The DNA remains as it is, even when the field is removed. , Since the parallel component of an electrostatic field is zero on a conducting surface, immobilization of stretched DNA onto a conducting surface requires a special method. Fig. 8 shows a “Fluid Integrated Circuit (FIC)” [ 101 device developed for this purpose. It consists of a pair of vacuum-evaporated aluminum electrodes, and a glass coverage which is open at one side. The electrodes are designed to: 1) create high intensity field at the gap, and 2) pass a dc current to generate a heat-induced flow. The procedure is as follows: First, the DNA sample is brought into the gap, and the electric field is applied. After the molecules are aligned at the electrode edge, a dc current is passed to the electrode. The heat generated by the current induces thermal expansion resulting in a flow toward the open end of the coverage. This flips the stretched DNA at the edge of the other electrode onto the conducting surface. As DNA tends to be adsorbed on an aluminum surface, the molecules flipped back and pressed against the A1 surface are fixed. Fig. 9 shows a photograph of DNA fixed on an aluminum surface with this technique.

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a) top view

Fig. 9. DNA immobilized on an aluminum surface.

15 floatingpotential electrodes b) cross sectional An application of this method is found in STM (Scanning Tunneling Microscopy) specimen preparation. There have been reports on the observation of nucleic acid molecules with STM [ 111-[13], but reproducibility in such observations is not necessarily high. A question often raised is whether c) enlarged &over SED the image obtained is the real DNA or some artifacts, such as line dislocations of the substrate lattice. This stems from qFubstrate-hJ the difficulty in locating the molecules in the conventional electrode electrode specimen preparation method, where nucleic acid solution Fig. 10. Floating potential electrode geometry for electrostatic immobilizais just dropped on a surface and dried. In contrast, the tion of DNA. method of Fig. 8 provides definite location (in contact with the electrode edge) and orientation (perpendicular to the edge). The flat conducting surface required in STM observations are to an electrode edge, but the other end is prevented from obtainable by vacuum evaporation on a cleaved mica. With the approaching to the other electrode by the flow. Therefore, we developed an electrode configuration which use of this method, a direct sequencing of genetic information with STM might be possible in the future when the resolution is less likely to induce flow than a simple parallel strip (see of the scanning microscopy reaches high enough to recognize Fig. IO). It consists of two outermost electrodes, and several thin strip electrodes between the gap. The power supply is the base sequences. connected only to the outermost electrodes, and the thin strip electrodes have no electrical connections (floating potential B. Site-Specific Immobilization electrodes). We call it “floating potential electrode geometry.” The electrodes of Fig. 10 are fabricated by etching a The role of DNA in life is the storage of genetic information. Its reading out is performed by an enzyme, RNA polymerase, vacuum-evaporated aluminum layer on a glass substrate. which mounts on a DNA and synthesizes RNA as it travels The spacing between the floating electrodes is made slightly along the strand. If the DNA is in contact with a substrate at smaller than the length of DNA to be immobilized. In the some point, steric hindrance may prevent farther locomotion present experiment using A DNA which is 16.2 pm long, four floating potential electrodes with 5 pm width are placed of the enzyme. In the DNA immobilization methods of the previous section, between the energizing electrodes with the spacing of 15 which point of a DNA is fixed and which part is free pm. As shown in the figure, the field lines constrict at the is unpredictable. In order to allow the readout of arbitrary conductor edges, and dielectrophoresis pulls DNA to this position, it is desirable that the molecule is anchored only at region until both molecular ends bridge over the floating both ends, and the portion between the ends remain without potential electrodes and are anchored. This electrode configuration does not reduce the electric contact to the substrate. This section presents methods to field-induced flow at the edge of the energized outermost realize such “site-specific’’ immobilization. Electrostatic Site-Specific Immobilization: As mentioned electrodes, but the flow at the edge of the floating electrodes above, we found that an electrostatically stretch-and-positioned are substantially suppressed. With this field geometry, DNA is DNA is firmly anchored to the edge of a vacuum-evaporated anchored at both ends, bridging over the two adjacent floating aluminum electrode. This fact suggests that if the gap between potential electrodes. Fig. 11 shows the photograph of thus the electrodes is made slightly smaller than the length of DNA, immobilized DNA stained with DAPI. Only three of the four a DNA can be fixed at both molecular ends bridging over floating potential electrodes are seen in the figure. The DNA the gap. However, this method was proved unsuccessful for in the photo is not perfectly straight but somewhat bent by the the reason as follows: There exists a flow of the medium flow of the medium of about 50 pm/s (the flow velocity is associated with the very high intensity field at the electrode measured by tracing a floating particle, and the velocity can edges. The flow pattem in the gap starts from both electrode be substantially lower near the substrate where DNA is fixed). edges toward the center of the gap, and goes apart from This indicates that the part other than both ends are free, and the plane of the electrodes, and circulates back. One end the fixing ends are strong enough to support the hydrodynamic of the DNA pulled into the high field region may be fixed drag all over the length of the molecule.

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Fig. 11. DNA immobilized on the floating potential electrodes.

The fact that the flow occurs only on those electrodes with electrical connections to the extemal power supply may suggest the relevance of charge injections. However, it is unclear whether a charge injection can result in a steady flow under our experimental conditions with a 1-MHz power supply or not. We have not finished a thorough investigation about its mechanisms, but just report that the floating potential geometry is effective for the anchoring of DNA at both its ends. It should be added that the floating potential electrode geometry can be used for the investigation of the binding of the DNA-ends on the electrode edge. The question is whether the very high intensity field at the sharp edge of the vacuum-evaporated electrodes, or the contact with the aluminum surface, can be the cause of any degradation of the DNA structure or not. To answer the question, immobilized DNA in the floating potential electrodes is treated with an exonuclease. The result is that most DNA molecules come off the electrodes. This means that the neither the binding itself, nor the possible degradation of DNA, can cause hindrance to the activity of the nuclease. The basic structure of DNA is preserved, although there may be damages to the bases. Immobilization Using Biotin-Avidin Binding: Potential problems in the previous immobilization method are that: 1) DNA can be fixed only on the electrode edge, and 2) the mechanism of DNA binding to the electrodes is unclear, so that the binding may cause steric hindrance for some molecular processes. Therefore, an altemative method of site-specific immobilization is developed, which enables the immobilization with a known molecular bonding onto virtually any surfaces. The method uses a strong molecular binding between avidin and biotin [I41 (affinity constant of 1015[M-1]).Biotin molecules are introduced to the 3‘ end of a DNA strand with a biochemical procedure [15]. Also, a substrate surface can be biotinated with the use of photo-linkable biotins. Because avidin is a tetramer, and thus accepts four biotin molecules per molecule. it can be used as the binding agent between the biotin at the edge of DNA and the biotin on the substrate. A stretch-and-positionedDNA molecule is subject to hydrodynamic drag when there is a bulk flow, as observed in Fig. 1 1. The longer the DNA is, the larger the stress at the anchored

Fig. 12. Schematic representation of the immobilization of an electrostatically stretch-and-positionedDNA onto silane-coupler coated surface using avidin-biotin binding. (Binding location of photobiotin on the silane coupler is not clear).

point becomes. In particular, the binding must endure the hydrodynamic stress during the exchange of the medium. We found experimentally that the avidin-biotin binding is strong enough but the weakest is the binding between biotin and the substrate. An immobilized DNA is often dissociated from this point. Therefore, we developed a surface pretreatment, which allows secure attachment of photobiotin molecules. The method is as follows: The surface treatment with silane couplers introduces covalently-linked carboxyl groups on the electrode/substrate. The negative charges on the carboxyl group prevent nonspecific adsorption of DNA. Then the substrate is treated with photo-linkable biotin (photoprobe biotin, Vector laboratories Inc.) to obtain a biotinated surface. On the other hand, the biotinated DNA solution is mixed with avidin to form a biotin-avidin complex at both ends of each molecule. Then the mixture is dialyzed against water to lower the conductivity. Finally it is brought into the electrode gap biotinated with the above process, and the field is applied. The DNA is stretched and aligned onto the electrode edge, and the avidin’s vacant binding sites at both ends of the DNA (there are 4 binding sites and only one is occupied by a biotin on the DNA) bind to biotin on the substrate. Fig. 12 shows schematically the binding of DNA onto the substrate expected with this method. It has been confirmed that DNA is actually anchored at both ends with this method, and the binding is strong enough to withstand the tension during the exchange of the medium, from the low-conductivity medium in which the electrostatic stretch-and-positioning takes place, to a physiological one in which biochemical procedures are conducted. An application of this technique is to the observation of the biophysical process of the transcription. The transcription is performed by the RNA polymerase, which synthesizes RNA as it moves along a DNA strand. The synthesis is known to start at a particular sequence, called a promoter, but there has been controversy in how an RNA polymerase first reaches the promoter [16]. One hypothesis is that the enzyme

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first nonspecifically binds to some portion of the DNA and slides along the strand, until it reaches the promoter. The motion is confined in one-dimension, and should be more efficient for searching promoter location than relying on a mere three-dimensional diffusion. The sliding of fluorescent-labeled RNA polymerase in fact is experimentally observed on the electrostatically immobilized DNA [ 171, [ 181. V. APPLICATIONS IN DNA SEQUENCING The most important application of the electrostatic stretchand-positioning method may be to the sequencing of nucleic acids. The spatial resolution provided by the physical manipulation of DNA is a great advantage over chemical methods. In the method previously proposed by the authors [2], the stretched DNA strands are cut into small pieces from one of its ends with ultraviolet (UV) beam and the fragments are successively subcloned and sequenced. In this way, the location of each fragment is known within the optical resolution, so that the reconstruction of the total sequence becomes much easier. Further refinement of the method has been developed, which is reported in this section.

Fluid Passage

Cover slip (a)

-

resin

Glass Substrate (b) Fig. 13. FIC device for the recovery of laser-cut DNA fragments

A. FIC Device for the Recovery of the Fragments

In the UV cutting method, the DNA is aligned on the surface of the substrate and scanned with a UV laser beam. The number of molecules handled by the laser cut may be of the order of lo3. This number is far smaller than that handled in conventional biochemistry: Present radioactive tracer methods permit measurements of DNA of the order of 100 pg, which represents 6 x lo9 molecules if the DNA size is 10 kD. However, the small number of DNA involved in the laser cut does not cause a serious problem by itself, because there are established techniques for DNA amplification such as the cloning or PCR. The problem is how the DNA fragments cut in the microstructured electrode gap can efficiently be recovered. Fig. 13 shows the FIC device developed for this purpose. It consists of a pair of vacuum-evaporated parallel strip electrodes and a fluid channel covering the electrode gap, with an inlet and an outlet. Electrodes and insulating surfaces inside the channel are surface-treated with fluorosilane to prevent adhesion of DNA. DNA solution is fed from the inlet, and the electrodes are energized to align DNA onto the electrode edge. The DNA molecules are anchored with the ends touching the electrode edge, despite that the edge itself is covered with fluorosilane. Then they are irradiated with the UV laser, and the resultant free fragments diffuse into the medium. Fig. 14 shows the cutting of DNA with an Nz laser (wavelength 337 nm), when the laser spot of 1 pm in diameter is scanned vertically in the figure for about 10 pm. The DNA molecules, aligned horizontally in the figure, are cut at the point of irradiation, so the strands remaining on the electrode are shorter than the original length. After the cut, the applied voltage should be removed as soon as possible in order to prevent the DEP attraction of the fragments to the electrode edge, which reduces the recovery yield. Thus, the fragments are released into the medium, while the remainder part is still fixed onto the electrode edge. The fragments are recovered

Fig. 14. DNA cut by laser beam in the FIC of Fig. 13.

at the outlet by a pressurization from the inlet, or a suction from the outlet. The binding of DNA to the electrode edge is experimentally found to be intact during this process. A serious technical problem may be that the DNA molecules are invisible under a microscope when the laser cut is performed. Binding of a fluorescent dye is often irreversible, and it will jeopardize later biochemical procedures. However, the reproducibility of the electrostatic stretch-and-positioning of DNA is so good that, if the DNA length is known, we have experienced no serious difficulty in blind-cutting at the predetermined location. The mechanism of the laser cutting of DNA is not clear to us. No enzymatic molecules are present when the laser cut is performed, so the mechanism should be a pure physical one. The photon energy of the Nz laser, 37 eV, is slightly larger than the binding energy of the C-C bond, and is able to cut virtually any biochemical bonds. It is conceivable that many of the bases in the DNA helix are destroyed before the DNA is cut and separated into two pieces. The optics limit the minimum laser spot size to be about its wavelength. This can cause the molecular damage in the area of about 1 kb around the point of irradiation. Whether or not the damaged part can be enzymatically tailored to be compatible with biochemical proceedings remains to be clarified.

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D U

U D U D D

fluolescentprobe

DNA flagment with a known sequence

(a) restriction site

Electrostatically&etch-and-positioned single-strandDNA with unknown sequence Fig. 16. Detection of a particular sequence in a DNA using a stretched single-strand DNA.

digestion is shortened by this length, which is considered as an indication that the directional orientation as Fig. 15 is realized. Its detailed analysis awaits the completion of the analysis of laser cut fragments. The choice of the restriction enzyme in this method must (C) Fig. 15. Principle of the unidirectional orientation of DNA using a restriction be made with trial-and-error in general, because we do not enzyme. (a) Bidirectional orientation of DNA achieved by the electrostatic know the sequence of the DNA beforehand. The role of the stretch-and-positioning.(b) A restriction enzyme, which cuts the DNA at a enzyme here is to cut the DNA near one end of its contour, location near U end is added. (c) DNA anchored to the electrode edge with so the DNA size measurement in Chapter I1 of this paper will U end is removed, and unidirectionally oriented DNA is obtained. provide a quick method for the choice of the enzyme. B. Directional Orientation

A double-stranded DNA molecule consists of two polynucleotides, which are antiparallel to each other. This means that the molecule is electrically symmetrical, so that the electrostatically stretch-and-positioned DNA is a mixture of two orientations. Therefore, the laser-cut fragments obtained by the method in the previous section are a two-component mixture. In order to obtain the fragments from a particular location only, the alignment of DNA in one orientation (directional orientation) is necessary. The discrimination between the two orientations requires a recognition of the base sequences. One possible way is to use a molecule that specifically binds to a particular sequence near one end of DNA: Pattem a thin line of the antibody against that molecule in the electrode gap, parallel to the electrode edge. Then pin DNA with the antigen-antibody reaction at this position, and stretch electrostatically. The alignment of DNA with a known orientation is possible with this method. An altemative is to remove one orientation from the mixture of the two orientations. Its principle is schematically illustrated in Fig. 15. The notations U and D in the figure are used to show the upstream and downstream side of a gene of interest. First, DNA is aligned, and fixed to the electrode edge with the method in the previous section. This gives the 50% mixture of the two orientations. Then a restriction enzyme is added, which cuts at a position near one end of the DNA, say near the U end. The DNA molecules fixed at the electrode with the U end are removed in this stage, while those fixed at the D end remain. Thus the unidirectional orientation becomes possible. This method of using a restriction enzyme has a practical advantage over the pinning method in that restriction enzymes with various restriction sites are commercially available. In our experiment, a six base cutter (Bbe I), which cuts a X DNA at 1 lLm from one of its ends, is used. The DNA after the enzymatic

VI. SOMEFUTURE APPLICATIONS The molecular manipulation of DNA enabled by an electrostatic field can potentially bring drastic changes in sequencing. For instance, when the resolution of scanning microscopy reaches real atomic level, the direct sequencing of stretch-andpositioned DNA may replace all the conventional sequencing methods. In the shorter term, an application may be found in the detection of a particular sequence on a DNA strand. It is conventionally done with a hybridization technique: The DNA sample is denatured (i.e., separated into two single strands), and single-stranded nucleic acid fragments with a know sequence, labeled with a radioactive or a fluorescent probe, are added. Formation of a hybrid among these two nucleic acids is the indication that the sequence, complementary to that of the added fragment, exists in the sample DNA. The existence of the sequence can be detected with this method; however, it does not provide information about its location. The stretch-and-positioning of single-stranded DNA followed by the hybridization with the fluorescent-labeled fragments (see Fig. 16) can provide a spatial resolution. Here, a problem is that a single-stranded nucleic acid is not stretched under an electric field, as previously mentioned. The solution to this problem is to fix both of the ends of a double-stranded DNA onto a substrate first, and then to enzymatically remove one of its strands. The site-specific immobilization techniques in Section IV of this paper can be used for this purpose. The investigations are presently ongoing toward this goal in our lab. VII. CONCLUSION

Based on the finding that DNA molecules are stretchand-positioned using 1 x lo6 V/m 1-MHz field created in microfabricated electrodes, its applications to biochemical

WASHIZU et al.: APPLICATIONS OF ELECTROSTATIC STRETCH-AND-POSITIONING OF DNA

assays and biophysical researches are studied. The results are summarized as: DNA size distribution is determined by measuring the density distribution of electrostatically stretch-andpositioned DNA as a function of the distance from the electrode edge. Nuclease activity, i.e., the speed at which DNA is digested, is measured by the length of electrostatically stretched DNA. Single-stranded DNA is not electrostatically stretched under a 1 x lo6 V/m, 1 MHz field. An FIC (Fluid Integrated Circuit) device is developed to realize stretch-and-positioning of DNA onto a conducting surface. Two site-specific immobilization methods of the electrostatically stretch-and-positioned DNA strands are developed, one using the floating potential electrode geometry, and the other using the avidin-biotin binding. Both methods realize anchoring of a DNA molecule only at its ends, leaving the center part without contact to the substrate. An FIC device is developed for an efficient recovery of laser-cut DNA fragments. A method to obtain unidirectional orientation of DNA is developed. has been shown that DNA can be anchored onto a substrate only at its ends, in such a way that transcription can take place. This may be considered as an example of controlled molecular manipulations where the biological function of a molecule is preserved. The electrostatic molecular manipulation enables physical observations, measurements, and operations of DNA, where the position of the molecule can be controlled, and the location in a molecule can be addressed. The method, using microfabricated electrode structures, requires a far smaller number of molecules compared with conventional chemistry. It is hoped that the method present in this paper may open a way to a novel category of “molecular biochemistry with spatial resolution.” ACKNOWLEDGMENT The authors would like to acknowledge Prof. Senichi Masuda of Fukui Institute of Technology, Prof. Yasuo Hotta of Nagoya University, Prof. T. B. Jones of Rochester University, Prof. R. Pethig of the University of Wales, Prof. A. Kat0 of Seikei University, and Dr. H. Nagai, H. Kabata, and T. Kubori of the National Institute of Genetics for valuable discussions.

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J. D. Watson, N. H. Hopkins, J. W. Roberts, J. A. Steitz and A. M. Weiner, Molecular Biology offhe Gene, 4th ed. Menlo Park, CA: The BenjaminKummings Publishing Co., 1987. R. W. Hendrix, J. W. Roberts, F. W. Stahl, and R. A. Weisberg, Eds., Lambda II. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1983. D. R. Brown, J. Hunvits, D. Reinberg, and S. L. Zipursky, ‘‘Nuclease activities involved in DNA replication,” in Nucleases, S. M. Linn and R. J. Roberts. Eds. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1985. S. Matsumoro, K. Morikawa, and M. Yanagida, “Light microscopic structure of DNA in solution studied by the 4’. 6-diamidino-2phenylindole staining method,” J. Mol. Biology, vol. 152, pp. 501-5 16, 1981. S. Masuda, M. Washizu, and T. Nanba, “Novel method of cell fusion in field constriction area in fluid integrated circuit,’’ IEEE Trans. Ind. Applicaf., vol. 25, no. 4, pp. 732-737, 1989. D. D. Dunlap and C. Bustamante, “Images of single-stranded nucleic acids by scanning tunneling microscopy,” Nature, vol. 342, pp. 204-206, 1989. G. Lee, P. G. Arscott, V. A. Bloomfield, and D. F. Evans, “Scanning tunneling microscopy of nucleic acids,” Science, vol. 244, pp. 475-477, 1989. T. P. Beebe, T. E. Wilson, D. F. Ogletree, J. E. Kats, R. Balhorn, M. B. Salmeron, and W. J. Siekhaus, “Direct observation of native DNA structures with the scanning tunneling microscope,” Science, vol. 243, pp. 37C372, 1989. N. M. Green, “Avidin,” in Advances in Protein Chemisfry, C. B. Anfinsen, J . T. Edsall and F. M. Richards, Eds. New York: Academic, 1975, vol. 29, pp. 85-133. M. Fujioka, T. Hirata, and N. Shimamoto, “Requirement for the +-j pyrophosphate bond of ATP in a stage between transcription initiation and elongation by E. coli RNA polymerase,” Biochemistry, vol. 30, pp. 1801-1807, 1991. P. von Hippel and 0. G. Berg, “Facilitated target location in biological systems,” J. Biol. Chem., vol. 262, pp, 675-678, 1989. N. Shimamoto, K. Kabata, 0. Kurosawa, and M. Washizu, “Visualization of single molecule of RNA polymerase and its application for detecting sliding motion on T7 DNA dielectrophoretically manipulated,” Sfrucfurul Tools for the Analysis of Protein-Nucleic Acid Complexes, D. Lilley, H. Heumann, and D. Suck, Eds. Basel, Birkhauser Verlag, 1992. H. Kabata, 0. Kurosawa, I. Arai, M. Washizu, S. A. Margason, R. E. Glass, and N. Shimamoto, “Visualization of single molecules of RNA polymerase sliding along DNA,” Science, vol. 262, pp. 1561-1563, 1993,

Masao Washizu (M’87) received the Ph.D. degree in electrical engineering from the University of Tokyo, Japan, in 1981. He was a research engineer at Toshiba Corporation from 1981 to 1985. He is currently an associate professor in the Department of Electrial Engineering and Electronics, Seikei University, Tokyo. His primary research interest is in the application of electrostatics to bioengineering, including the electrostatic manipulations of biological objects using micromacbined structures. Dr. Washizu is a member of the Institute of Electrostatics Japan and the Institute of Electrical Engineers of Japan.

REFERENCES 111 J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1989. L21 M. Washizu and 0. Kurosawa, “Electrostatic manipulation of DNA in microfabricated structures,” IEEE Trans. Ind. Applicat., vol. 26, no. 6, pp. 1165-1 172, 1990. H. A. Pohl, Dielectrophoresis. Cambridge, UK: Cambridge University 131 Press, 1978. 141 R. Pethig, Dielectric and Electronic Properties qf Biological Materials. New York: Wiley, 1979. 151 D. Rickwood and B. D. Hames, Eds., Gel Electrophoresis of Nucleic Acid.y. Oxford: IRL Press, Oxford University Press, 1990.

Osamu Kurosawa received the B.S. degree from the Department of Biology, Waseda University, Japan, in 1987. He joined Advance Company, Tokyo, Japan, in 1987, where he is presently a Research Scientist. His research area is in the electrostatic manipulation of biological molecules.

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Ichiro Arai received the M.S. degree from the Department of Electrical Engineering and Electronics, Seikei University, Tokyo, Japan, in 1994. He is currently employed with the Technology Development Department, Nikon Corporation, Tokyo.

Seiichi Suzuki received the Ph.D. degree in medical electronics from the University of Tokyo, Japan. He is currently a research associate in the Department of Electrical Engineering and Electronics, Seikei University, Tokyo. His primary research interest is in the protein function in relation with other propertieh, such as structure, mobility, and dielectric constant.

Nobuo Shimamotn received the Ph.D. degree from

Kyoto University, Japan, in 1977, where he studied the catalytic mechanism of chymotrypsin. He worked as a research associate with Dr. C.W. Wu, at the Albert Einstein College of Medicine, where he studied the mechanism of RNA synthesis by E. coli RNA polymerase. He joined the Faculty of Integrated Arts and Sciences, Hiroshima University, Japan, in 1979, as an assistant. He then joined the DNA Research Center, National Institute of Genetics. Mishima, Japan, as an associate professor in 1989. He is interested in the dynamic feature of gene expression and its regulations, carrying out single-molecule dynamics, and DNA-protein interaction and kinetic study on transcription by using immobilized template DNA.