Single-Molecular AFM Probing of Specific DNA Sequencing Using

Sequencing Using RecA-Promoted Homologous. Pairing and Strand Exchange. Gi Hun Seong, Tomohisa Niimi, Yasuko Yanagida, Eiry Kobatake, and Masuo ...
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Anal. Chem. 2000, 72, 1288-1293

Single-Molecular AFM Probing of Specific DNA Sequencing Using RecA-Promoted Homologous Pairing and Strand Exchange Gi Hun Seong, Tomohisa Niimi, Yasuko Yanagida, Eiry Kobatake, and Masuo Aizawa*

Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

The specific sequence in a linearlized double-stranded DNA target has been identified at a single-molecular level by atomic force microscopy (AFM). This was accomplished using RecA-coated, single-stranded DNA probes which were paired with a specific complementary DNA sequence in a linear double-stranded DNA target by strand-exchange reaction at a homologous sequence site with target DNA. The sites of interaction between the nucleoprotein filaments and the double-stranded DNA targets were directly visualized by AFM in solution containing 4 mM magnesium acetate. Measurements of the position of RecA-coated probes paired to individual target DNA showed that DNA probes specifically paired at their corresponding homologous target sequences. Strand exchange promoted by RecA and the visualization by AFM provided a rapid and efficient way to identify homologous sequence on a single-molecule target DNA. RecA from Escherichia coli binds to single-stranded DNA in the 5'- to 3′-direction1 and forms an extended right-handed helix in the presence of ribonucleoside triphosphate cofactors such as ATP or ATPγS,2,3 which play a key role in genetic recombination, DNA repair, and UV-induced mutagenesis.4 It efficiently coats single-stranded DNA oligonucleotides to form nucleoprotein filaments and catalyzes strand exchange between DNA molecules sharing regions of homology and target double-stranded DNA sequences.5-7 RecA-coated single-stranded probes can form stable joint molecules with homologous double-strand sequences on the target DNA molecule.8-10 * To whom correspondence should be addressed: (tel) +81-45-924-5759; (fax) +81-45-924-5779; (e-mail) [email protected]. (1) Register, J. C. III; Griffith, J. J. Biol. Chem. 1985, 260, 12308-12312. (2) Cox, M. M.; Lehman, I. R. Annu. Rev. Biochem. 1987, 56, 229-262. (3) Radding, C. M. Biochim. Biophys. Acta 1989, 1008, 131-145. (4) Walker, G. C. Annu. Rev. Biochem. 1985, 54, 425-458. (5) Shane, S. L.; Flory, J.; Radding, C. M. J. Biol. Chem. 1987, 262, 92209230. (6) Flory, J.; Tsang, S. S.; Muniyappa, K. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 7026-7030. (7) Williams, R. C.; Spengler, S. J. J. Mol. Biol. 1986, 187, 109-118. (8) Rao, B. J.; Dutreix, M.; Radding, C. M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2984-2988. (9) Lindsley, J. E.; Cox, M. M. J. Biol. Chem. 1990, 265, 10164-10171. (10) Jain, S. K.; Inman, R. B.; Cox, M. M. J. Biol. Chem. 1992, 267, 4215-4222.

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For identifying the protein-binding sites on DNA, biological methods such as the electrophoretic mobility shift assay (EMSA)11 and DNase footprinting analysis have been currently used.12 However, these techniques are not suited, because they are effective in analyzing the narrow range of a DNA sequence in a single experiment. Local atomic structures of DNA-protein complexes have also been studied using a variety of biophysical techniques, such as NMR and X-ray crystallography.13-15 These conventional imaging techniques, however, can provide information about higher-order structures of only short DNA-protein complexes and require at least two-dimensional crystals for analysis. An alternative to identifying protein-binding sites on DNA is to use microscopy to visualize protein binding on a singlemolecular DNA. Enzymes such as RNA polymerase and other proteins bound to DNA have been imaged by atomic force microscopy (AFM).16,17 Recently, a restriction enzyme site map was constructed by AFM using a mutant endonuclease.18 Also, a site-specific antibody for a Z-DNA sequence bound to a plasmid19 and sequence-specific interactions of triple-helix-forming oligonucleotides (TFOs) with double-stranded DNA were visualized by AFM.20 Since its invention by Binning et al. in 1986, AFM is emerging as a versatile tool for allowing the visualization of proteins, DNA, RNA, and protein-nucleic acid complexes at a nanoscale in the absence of stains, shadow, and labels.21-32 Particularly, with the (11) Lane, D.; Prentki, P.; Chandler, M. Microbiol. Rev. 1992, 56, 509-528. (12) Tullius, T. D. Annu. Rev. Biophys. Biophys. Chem. 1989, 18, 213-237. (13) Ellenberger, T. E.; Brandl, C. J.; Struhl, K.; Harrison, S. C. Cell 1992, 71, 1223-1237. (14) Hegde, R. S.; Grossman, S. R.; Laimins, L. A.; Sigler, P. B. Nature 1992, 359, 505-512. (15) Schleif, R. Science 1988, 241, 1182-1187. (16) Bustamante, C.; Vesenka, J.; Tang, C. L.; Rees, W.; Guthold, M.; Keller, R. Biochemistry 1992, 31, 22-26. (17) Hansma, H. G.; Bezanilla, H.; Zenhausern, F.; Adrian, M.; Sinsheimer, R. L. Nucleic Acids Res. 1993, 21, 505-512. (18) Allison, D. P.; Kerper, P. S.; Doktycz, M. J.; Spain, J. A.; Modrich, P.; Larimer, F. W.; Thundat, T.; Warmack, R. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8826-8829. (19) Pietrasanta, L. I.; Schaper, A.; Jovin, T. H. Nucleic Acids Res. 1994, 22, 3288-3292. (20) Cherny, D. I.; Fourcade, A.; Svinarchuk, F.; Nielsen, P. E.; Malvy, C.; Delain, E. Biophys. J. 1998, 74, 1015-1023. (21) Hansma, H. G.; Vesenka, J.; Siegerist, C.; Kelderman, G.; Morrett, H.; Sinsheimer, R. L.; Bustamante, C.; Elings, V.; Hansma, P. K. Science 1992, 256, 1180-1184. 10.1021/ac990893h CCC: $19.00

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ability to image specimens in solution,21-24 AFM is uniquely suited for biological studies and provides a means by which the macromolecular interactions responsible for genetic and cellular regulation can be characterized under native conditions. AFM observation in solution has some advantages over electron microscopy (EM); i.e, AFM can image under well-specified ionic conditions of sample without evaporation of water during the sample preparation, unlike EM,21-25 and visualize specimens without rapid change of buffer condition during drying of the sample followed by deposition of the sample. Also, complexes of DNA with sequence-specific bound ligands can be directly detected by AFM via their coupling with small proteins that cannot be seen directly in EM.20,29 In this work, we attempted to visualize, by imaging in solution using AFM, DNA-protein complexes formed by the pairing of RecA-coated complementary short single-stranded DNA probes with linear double-stranded DNA targets and to identify the sequence-specific site complementary to the single-stranded DNA probe on the double-stranded DNA target. Also, it was demonstrated that direct AFM imaging and the use of RecA as a reagent in searching for the homologous sequence provide an efficient way to allow homologous sequence positioning and gene mapping on individual double-stranded DNA. EXPERIMENTAL SECTION Materials. Plasmid pBluescript II SK- (2961 bp, from Stratagene, La Jolla, CA) in E. coli strain JM109 was purified and linearized by digestion with ScaI restriction enzyme as presented in Figure 1. The digested DNA was deproteinized by phenol/ chloroform extraction, followed by gel band isolation, and served as the target DNA. Probe DNA was synthesized (espec-oligo, Japan) and was resuspended in MilliQ water. The probe DNA (36-base oligonucleotide) had the sequence 5′-TCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATC-3′ and contained recognition site (italic; GAATTC) for EcoRI restriction enzyme. RecA (2.58 µg/µL) in buffer containing 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, and 50% glycerol was purchased from Promega (Madison, Wisconsin). A 100 mM solution of ATPγS (Boehringer Mannheim, Germany) was diluted to 10 mM just before use. Oligo(dT) (12-18-base oligonucleotide) was purchased from Pharmacia Biotech (Uppsala, Sweden). (22) Lyubchenko, Y. L.; Shlyakhtenko, L. S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 496-501. (23) Kasas, S.; Thomson, N. H.; Smith, B. L.; Hansma, H. G.; Zhu, X.; Guthold, M.; Bustamante, C.; Koo, E. T.; Kashlev, M.; Hansma, P. K. Biochemistry 1997, 36, 461-468. (24) Guthold, M.; Bezanilla, M.; Erie, D. A.; Jenkins, B.; Hansma, H. G.; Bustamante, C. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12927-12931. (25) Hansma, H. G.; Laney, D. E.; Bezanilla, M.; Sinsheimer, R. L.; Hansma, P. K. Biophys. J. 1995, 68, 672-677. (26) Hansma, H. G.; Sinsheimer, R. L.; Li, H.-Q.; Hansma, P. K. Nucleic Acids Res. 1992, 20, 3585-3590. (27) Lyubchenko, Y. L.; Shlyakhtenko, L. S.; Harrington, R. E.; Oden, P. I.; Lindsay, S. M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2137-2140. (28) Wyman, C.; Rombel, I.; North, A. K.; Bustamante, C.; Kustu, S. Science 1997, 275, 1658-1661. (29) Wyman, C.; Grotkopp, E.; Bustamante, C.; Nelson, H. C. M. EMBO J. 1995, 14, 117-123. (30) Rees, W. A.; Keller, R. W.; Vesenka, J. P.; Yang, G.; Bustamante, C. Science 1993, 260, 1646-1649. (31) Becker, J. C.; Nikroo, A.; Brabletz, T.; Reisfeld, R. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9727-9731. (32) Cary, R. B.; Peterson, S. R.; Wang, J.; Bear, D. G.; Bradbury, E. M.; Chen, D. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4267-4272.

Figure 1. Schematic diagram of ScaI-linearized plasmid pBluescript II SK- (2961 bp in size) used as double-stranded DNA target. The 36 bp targeting site homologous with 36-mer single-stranded DNA probe is located 1120 and 1805 bp from both the ScaI-cut ends and one EcoRI site is centered at the 36 bp targeting site.

Preparation of RecA-Target DNA Complex. Nucleoprotein filaments were first formed by incubating with 1 µL of 68.2 µM RecA (2.58 µg/µL) and 3 µL of 1.74 µM probe DNA (20 ng/µL) at 37 °C in a buffer consisting of 25 mM Tris-acetate (pH 7.5) and 1 mM magnesium acetate. After 1 min, 1 µL of 10 mM ATPγS and 1 µL of 17.6 µM oligo(dT) (80 ng/µL) were added to bring the final volume to 10 µL, and the reaction proceeded at 37 °C for 10 min. Linearized double-stranded DNA targets (80 ng) were added, the buffer was adjusted to 25 mM Tris-acetate (pH 7.5), 4 mM magnesium acetate, 10 mM dithiothreitol, and 2.7 µL of BSA (0.1 mg/mL), and incubation was continued at 37 °C for 30 min. The final target reaction volume was 27 µL. To visualize RecADNA complexes by AFM, the samples were purified by incubating with 200 µL of StrataClean resin (Stratagene) for 30 min and centrifuging at 3500 rpm, immediately adjusting the magnesium acetate to a final concentration of 4 mM. Also, to identify the sequence-specific site protection of target DNA by nucleoprotein filaments, 15 units of EcoRI enzyme, 5 µL of 80 mM magnesium acetate, and 5 µL of 250 mM potassium acetate were added and the reaction was continued at 37 °C for 1 h (50 µL reaction volume). The solution of reaction was analyzed on 1% agarose gel with ethidium bromide. AFM Imaging. The NanoScope III MultiMode system (Digital Instruments, Santa Barbara, CA) operated in the tapping mode was used for these experiments. To immobilize samples, a mica plate activated with (3-aminopropyl)triethoxysilane (APTES; APmica)22,27 was used as a substrate. An oxide sharpened silicon nitride tip, mounted on a V-shaped cantilever, of length 120 µm (spring constant, 0.58 N/m) were employed for imaging in solution. They were mounted on a glass cell tip holder for the tapping mode, and the tip was made to manually approach the AP-mica surface mounted on the sample stage by use of an optical microscope. Approach was stopped when the distance between the tip and surface was 50-60 µm. The 20 µL of filtered DNA solution was injected directly by syringe needle into a small gap between the mica surface and the tip holder, and the tip was brought into tapping range under control of the instrument. The scanning parameters were as follows: frequency, 8.5-9.5 kHz; scanning rate, 1.45 Hz; number of samples, 512 × 512. The image was taken in the topographic mode. RESULTS Optimization of RecA-DNA Complex Formation. As schematically represented in Figure 2, RecA, which is composed of Analytical Chemistry, Vol. 72, No. 6, March 15, 2000

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Figure 3. Titration reaction to maximize the specificity and efficiency of RecA-mediated homologous DNA sequence recognition. To optimize the ratio of RecA to probe DNA, nucleoprotein filaments were formed with a constant amount of RecA (68.2 pmol) and an increasing amount of a synthetic 36-mer single-stranded DNA probe (0, 1.74, 3.48, 5.22, 6.96, and 8.70 pmol in lanes 2-7, respectively. Lane 1, marker). After incubating nucleoprotein filaments with linearized double-stranded DNA target at 37 °C for 30 min, EcoRI restriction enzyme was added and the reaction was allowed to proceed for 1 h. The sample was analyzed by agarose gel electrophoresis on a 1% agarose gel.

Figure 2. A schematic illustration of homologous pairing and strand exchange promoted by RecA acting on the model substrates, singlestranded DNA probe and linear double-stranded DNA target.

352 amino acids and has a molecular weight of 37 842, binds a single-stranded DNA probe to form a nucleoprotein filament in the presence of a linear double-stranded DNA target and the nucleoprotein filament is fixed specifically at the homologous DNA sequence. The reactions may be divided into a number of steps (presynapsis, synapsis, and strand exchange): (1) In the presynaptic phase, which involves the stoichiometric binding of RecA to single-stranded DNA, a nucleoprotein complex, which the single-stranded DNA lies within the RecA filament, is formed.33,34 (2) In the synaptic phase, the nucleoprotein complexes interact with double-stranded DNA, leading to the establishment of homologous contacts. Pairing is thought to occur by multiple random contacts,35,36 which in vitro are facilitated by the formation of large DNA aggregates that serve to concentrate the DNA.37 (3) The next reaction proceeds into the strand-exchange phase, in which strands are exchanged to form heteroduplex DNA.38-40 Strand exchange initiated as the 3′-end of the complementary (33) Wesa, S. C.; Cassuto, E.; Mursalim, J.; Howard-Flanders, P. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 2569-2573. (34) Egelman, E. H.; Yu, X. Science 1989, 245, 404-407. (35) Gonda, D. K.; Radding, C. M. Cell 1983, 34, 647-654. (36) Gonda, D. K.; Radding, C. M. J. Biol. Chem. 1986, 261, 13087-13096. (37) Tsang, S. S.; Chow, S. A.; Radding, C. M. Biochemistry 1985, 24, 32263232. (38) DasGupta, C.; Shibata, T.; Cunningham, R. P.; Radding, C. M. Cell 1980, 22, 437-446. (39) Cox, M. M.; Lehman, I. R. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 34333437. (40) West, S. C.; Cassuto, E.; Howard-Flanders, P. Proc. Natl. Acad. Sci. U.S.A. 1981, 178, 2100-2104.

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strand of the linear double-stranded DNA is transferred to the single-stranded DNA. During the course of the reaction, large amounts of ATP are hydrolyzed by RecA.41 To optimize the specificity and efficiency of RecA-mediated specific sequence recognition and protection and to eliminate the nonspecific protection caused by interacting with double-stranded DNA,42-44 a constant amount of RecA was titrated with an increasing amount of a synthetic 36-mer single-stranded DNA probe, following by digestion with EcoRI restriction enzymes. Figure 3 shows the result of the titration reaction. When the molar ratio of RecA to single-stranded DNA probe was ∼13, the EcoRI site in the target sequence was protected efficiently from EcoRI restriction enzymes without nonspecific binding by RecA (Figure 3, lane 5). When less than the optimal amount of probe DNA was used, the target DNA was protected nonspecifically by the excess RecA43 (Figure 3, lanes 2 and 3). Figure 4 shows the AFM image of RecA binding nonspecifically at target DNA. When excess probe DNA was used, the efficiency of protection was significantly reduced (Figure 3, lanes 6 and 7), most likely as a result of incomplete nucleoprotein filament formation. After nucleoprotein filaments were formed under the optimal amount of probe DNA, oligo(dT) was incubated to remove completely unpolymerized RecA before adding the target DNA. The nucleoprotein filaments were bound to the recognition site and the EcoRI restriction enzymes would not cleave the target DNA (Figure 5, lane 4). When the RecA component was omitted from the reaction, the EcoRI site centered in the recognition site of target DNA from 686 to 721 was cut completely by adding EcoRI restriction enzymes (Figure 5, lane 3). AFM Imaging of RecA-DNA Complexes. To immobilize RecA-DNA complexes for detection by AFM, we used AP-mica, which was obtained by treatment of freshly cleaved mica with (3aminopropyl)triethoxysilane to attach positively charged amino (41) Kowalczykowski, S. C. Annu. Rev. Biophys. Biophys. Chem. 1991, 20, 539575. (42) Ferrin, L. J.; Camerini-Otero, R. D. Science 1991, 254, 1494-1497. (43) Koob, M.; Burkiewicz, A.; Kur+, J.; Szybalski, W. Nucleic Acids Res. 1992, 20, 5831-5836. (44) Ferrin, L. J.; Camerini-Otero, R. D. Nature Genet. 1994, 6, 379-383.

Figure 4. AFM image of double-stranded DNA target bound nonspecifically by excess RecA.

Figure 5. Specific-sequence site protection from EcoRI endonuclease by RecA. Lane 1, marker for size control; lane 2, palsmid pBluescript II SK- linearized by ScaI restriction enzyme; lane 3, RecA among all the components in the reaction solution was only omitted; lane 4, the nucleoprotein filaments formed at the opimal ratio of RecA to probe DNA were bound with target DNA molecules.

groups to the mica surface.22,27,45 AP-mica prepared at a very low concentration of chemical in vapor and at ambient conditions permitted us to obtain a smooth and flat surface that can be used as a substrate for AFM studies of DNA. Before RecA-DNA complexes were observed under AFM, a solution of DNA was filtered through StrataClean resin, immediately adjusting the magnesium acetate to a final concentration of 4 mM. After filtering, proteins such as BSA and RecA in the DNA solution were almost removed (data not shown). A solution of purified DNA was injected into a glass cell, and RecA-DNA complexes bound to the surface were imaged in solution without drying the sample. Technically, the procedure of AFM imaging in air is much simpler than that in solution. However, by the evaporation of water during the sample preparation, the ionic condition of the vitrified sample is not well specified and DNA may change its conformation during the steps followed by the deposition of the sample. In this study, the concentration of magnesium acetate in a DNA buffer solution was kept constant at 4 mM during the AFM imaging. (45) Lyubchenko, Y. L.; Jacobs, B. L.; Lindsay, S. M.; Stasiak, A. Scanning Microsc. 1995, 9, 705-727.

Figure 6. Large-scale AFM image of linearized double-stranded DNA target paired specifically with RecA-coated, nucleoprotein filaments (arrow). Images were acquired using a tapping mode in buffer solution retaining magnesium acetate of 4 mM. The target site homologous with probe DNA is located 1120 and 1805 bp from both ends of 2961 bp target DNA. One DNA molecule, which is rarely found, the nucleoprotein filament is not site specifically bound (inset).

Figure 6 shows a typical large-scale AFM image of RecADNA complex in buffer solution. On the basis of a 0.34 nm baseto-base distance in B-form DNA,46 the predicted contour length of 2961 bp DNA is 1007 nm. We estimated the end-to-end distance of the DNA molecule to be 1031 ( 25 nm, 0.348 ( 0.008 nm (mean ( SD, N ) 50) base-to-base distance, ∼102% of the predicted contour length. Most of DNA molecules are not as extended and tend to be curved or even convoluted. In such cases, the better measurements of contour length can be obtained from a set of higher resolution images of contiguous regions of the molecule. An example of them is the zoomed image. From a complete set of such images, the contour length of the DNA molecule is able to be estimated. The width of DNA molecules determined from the different cross sections of images was between 4.2 and 6.3 nm (N ) 50). The crystallographic width of B-DNA is 2 nm, but the widening of DNA due to a finite size of the AFM probes is inevitable. From previous AFM studies where the probe tip was modeled as a hemisphere with radius R and the DNA as a cylinder with radius r, the expected width of DNA imaged by AFM can be calculated as 4(Rr)1/2.47 Another characteristic of DNA, the height of the DNA strands, was between 1.1 and 1.4 nm (N ) 50). The position of the probe on the target was determined by measuring the two parts of the target DNA that were not covered by the RecA-coated probe DNA. We obtained the following values, 378 ( 11 and 632 ( 15 nm (mean ( SD, N ) 15), which are in close agreement with the expected 1120 and 1805 bp values. Also, linearized double-stranded DNA targets paired specifically with (46) Watson, J. D.; Hopkins, N. H.; Roberts, J. W.; Seitz, J. A.; Weiner, A. M. Molecular Biology of the Gene, 4th ed.; Benjamin/Cummings: Menlo Park, CA; 1987; p 249. (47) Thundat, T.; Zheng, X.-Z.; Sharp, S. L.; Allison, D. P.; Warmack, R. J.; Joy, D. C.; Ferrell, T. L. Scanning Microsc. 1992, 6, 903-910.

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RecA-coated, nucleoprotein filaments were less than 30%. The inset image of Figure 6 shows the RecA-DNA complex bound to the nonspecific site on the double-stranded DNA target. However, almost all the plasmids used in this study were consistent with respect to molecular length and specific-sequence site binding. In previous studies by electron microscopy, the nucleoprotein filaments formed on either single-stranded or double-stranded DNA in the presence of ATPγS or ATP showed the DNA to be stretched to 150% the length of native B-form double-stranded DNA48-50 and the width of the filaments formed on single-stranded DNA and double-stranded DNA were 9.3 and 11.5 nm in diameter, respectively.6,7,51 Figure 7 shows a high-resolution image of a RecADNA complex obtained by scanning over a small area. In our AFM images, the length of the nucleoprotein filaments was 16.2 nm, which was longer than 12.4 nm for the predicted length of 36 base pair DNA. The width of triplex DNA nucleoprotein filament formed by strand exchange was ∼15 nm, wider than those of single- and double-stranded DNA. DISCUSSION During the last decade, numerous attempts have been made to use oligonucleotide, particularly TFOs,54,55 and lately, peptide nucleic acids (PNAs)56,57 as tools for exploring DNA structure and for creation methods for regulation of gene expression and genome analysis. TFOs and PNAs form triplexes with DNA, albeit of a different nature. However, the stability of the triplexes and the efficiency of their formation are significantly affected by (1) the nature and concentration of mono- and divalent cations, pH, and temperature, (2) the triplex structure and base composition, and (3) the length of the ligand. Recently, specific-sequence binding protein19 and a mutant endonuclease that binds its DNA recognition sequence but does not cleave DNA were used to map the binding site by AFM,18 but these methods have the limitation of being able to map only limited sequence sites. However, RecA-coated oligonucleotide probes form stable three-stranded DNA with a homologous DNA sequence located at any position on the target molecule, as long as the probe remains RecA-coated.8-10 Also, use of RecA to mediate the DNA pairing reaction between complementary single-stranded DNA probes and double-stranded DNA targets allows rapid homologous pairing without the need for denaturation of the target DNA and the hybridization with individual double-stranded DNA targets with high efficiency and precision. These properties of RecA suggest that RecA might be a useful reagent in searching for a homologous sequence. Also, the RecA-mediated pairing reaction between short singlestranded DNA probes and double-stranded DNA targets has many (48) Stasiak, A.; DiCapua, E.; Koller, T. J. Mol. Biol. 1981, 151, 557-564. (49) Dunn, K.; Chrysogelos, S.; Griffith, J. Cell 1982, 28, 757-765. (50) Flory, J.; Radding, C. M. Cell 1982, 28, 747-756. (51) Lindsley, J. E.; Cox, M. M. J. Mol. Biol. 1989, 205, 695-711. (52) Rigas, B.; Welcher, A.; Ward, D.; Weisman, S. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 9591-9595. (53) Sena, E. P.; Zarling, D. A. Nature Genet. 1993, 3, 365-372. (54) Beal, P. A.; Dervan, P. B. Science 1991, 251, 1360-1363. (55) Thuong, N. T.; Helene, D. Angew. Chem., Int. Ed. Engl. 1993, 32, 666690. (56) Knudsen, H.; Nielsen, P. E. Nucleic Acids Res. 1996, 24, 494-500. (57) Nielsen, P. E.; Egholm, M.; Berg, R. M.; Buchardt, O. Nucleic Acids Res. 1993, 21, 197-200.

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Figure 7. Small-scale AFM image of nucleoprotein filament paired to target DNA molecule. (A) Cross-sectional image to measure the length of a nucleoprotein filament; (B) topography image obtained by small-area scanning. (C) surface plot of the AFM image of nucleoprotein filament-target DNA complex.

potential in vitro applications. Biotinylated probes can be used in RecA-mediated pairing reactions to selectively isolate homologous target DNA molecules using affinity-selection techniques.52,53 The RecA catalyzes the formation of stable sequence-specific DNAprotein triplexes which protect the DNA from the activity of restriction enzymes and DNA methylases.42-44 An AFM-based approach described here is potentially applicable to identifying protein-DNA complexes and elucidating the structure-function relationship in DNA and protein interactions. Particularly, when constructing maps of specific-sequence binding sites on DNA, AFM enables the use of large-scale DNA templates. Using large-size DNA segments not only increases

efficiency in constructing specific-sequence binding maps but may also enable detection of long-range interactions such as enhancerpromoter interactions that are undetectable on short DNA fragments. The application of AFM in situ without any modification or crystallization of biological objects is expected to provide great potential to biological studies.

visual evidence that nucleoprotein filament binds at a specific sequence of the double-stranded DNA traget. The use of RecA as a reagent in searching for a homologous sequence and the visualization by AFM provided a rapid and efficient way to identify RecA-DNA filament binding site on the double-stranded DNA target.

CONCLUSION The results presented here show that AFM is used to investigate biological structures in aqueous environments at a single-molecular level. This instrument made it possible to obtain

Received for review August 5, 1999. Accepted December 9, 1999. AC990893H

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