Scanning force microscopy of nucleic acid complexes

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[ 1 1 ] Scanning Force Microscopy of Nucleic Acid Complexes By PETER T. LILLEHEIand LAWRENCE A.

BOTTOMLEY

Introduction Small ligands bind to DNA by (1) intercalation between adjacent base pairs, (2) binding within the major or minor grooves, (3) nonclassic modes] or (4) a combination of these modes. Definitive assays for mode of binding are threedimensional (3D) structure determination by X-ray diffraction or nuclear magnetic resonance (NMR) spectroscopy. These labor-intensive techniques are applicable only to binding studies of short DNA fragments because of difficulties in obtaining diffraction-quality crystals or interpretation of complicated chemical shift data. Their use is often precluded by lack of site specificity, rapid exchange, or multiple binding modes. Assays suitable for long DNA fragments involve viscometry, sedimentation, and linear and circular dichroism. These methods are reliable when ligands bind by conventional intercalative or minor groove modes, 2 but can be confounded by mixed and nonclassic modes. All traditional assays require large quantities of material. Whereas traditional techniques average over population and time, microscopic imaging techniques enable structural determinations of complexes from single molecules that are effectively frozen in time and space. Evidence of the mode and site of drug binding is, in principle, directly attainable from image analysis of a small number of nucleic acid molecules. Optical microscopy has insufficient spatial resolution for visualization of most drug-DNA complexes. The elaborate sample preparation techniques required for electron microscopy have precluded its use as an assay for drug binding. Scanning force microscopy (SFM, also known as atomic force microscopy) enables direct visualization of single DNA strands. A tip, fabricated onto the end of a readily deformable, rectangular or triangular-shaped cantilever, is positioned near or on the surface of the sample to be imaged. The tip and sample are moved with respect to one another and the cantilever bends in response to surface topography. Laser light is reflected off the cantilever onto a position-sensitive photodetector to track cantilever deflection. An image is produced by plotting cantilever deflection as a function of the position of the tip on the sample. SFM has advantages over optical and electron microscopy for imaging nucleic acids. Images can be acquired in vacuum, under liquid, or in air at ambient, 1 L. A. Lipscomb, K X. Zhou, S. R. Presnell, R. J. Woo, M. E. Peek, R. R. Plaskon, and L. D. Williams, Biothemistry 35, 2818 (1996). 2 D. Suh and J. B. Chaires, Bioorg. MeN. Chem. 3, 723 (1995).

METHODSIN ENZYMOLOGY,VOL.340

Copyright@ 2001 by AcademicPress All rightsof reproduclion in any lbrm reserved. 0076-6879/00 $35.00

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cryogenic, or elevated temperatures. Sample preparation is minimal. Addition of a stain or contrast agent, replica formation, or application of a conductive coating is not required. As a result, SFM is inherently a higher resolution technique. SFM has limitations. Images of nucleic acids can be obtained only when the molecule is physisorbed or chemisorbed to an atomically flat substrate. Image features are dependent on tip geometry and structural details are limited only to the portions of the molecule in direct interaction with the tip. Analysis and interpretation of images of nucleic acids require (1) assessment of tip shape and diameter as well as how these parameters may have changed during the course of imaging, (2) consideration of elastic and inelastic sample deformation, and (3) knowledge of the sources and treatment of image artifacts. Even with these limitations, the capability of acquiring subnanometer scale images of single molecules in vitro renders SFM superior to other forms of microscopy for determining nucleic acid structures.

Instrumental Considerations Imaging Modes Three modes are used to image nucleic acids: contact, noncontact, and intermittent contact imaging. Schematics of each are given in Fig. 1. Each has advantages and limitations. Contact Mode. As the name implies, with the contact imaging mode the tip is kept in contact with the sample. Contact mode images are obtained by plotting cantilever deflection as a function of the xy coordinates of the tip on the sample. Distortion of soft surfaces (or compliant molecules adsorbed on them) is commonplace with this imaging mode because of variation in the vertical force exerted by the tip on the sample. To image under fixed vertical load, a feedback mechanism is required. As the cantilever bends in response to surface topography, the scanner is retracted or extended to return cantilever deflection to its original value. Images are obtained by plotting the z piezovoltage as a function of the xy coordinates of the tip on the sample. Because the movements of the scanner are calibrated to subangstrom dimensions, accurate topographic images are obtained with properly tuned scanner feedback control. High-quality images of DNA can be acquired in just a few minutes with this imaging mode. Minimal vertical loading must be used to eliminate sample deformation or damage and tip wear. In the absence of strong adsorbate-substrate interaction, molecules will be readily displaced with this imaging method. When samples are imaged in air under ambient humidity, a thin film of water forms on the sample and tip surface. Contact between these water layers produces increased vertical and lateral forces that cause severe artifacts in the image. 3 Whenever possible, contact mode imaging should either be performed under a dry atmosphere or under fluid.

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a

I

11 11

%

FIG. 1. Schematic of the tip-surface interaction during (a) contact mode, (b) noncontact mode, and, (c) intermittent contact mode imaging.

Noncontact Mode. The noncontact imaging mode maintains the sample and probe tip in close proximity while scanning. The cantilever oscillates at its resonant frequency and any change in the forces acting on the cantilever (magnetic, electrostatic, and/or van der Waals forces) will be manifested as a shift in resonant frequency. Topographical information is obtained by monitoring resonant frequency shifts as a function of the xy coordinates of the tip above the sample. Tip wear and sample deformation or damage are eliminated in noncontact mode imaging. Because the forces are often small, tip-sample separation must be minimized. This mode is best suited for imaging under vacuum, in which van der Waals interactions are not dampened by intervening liquid or by adsorbed gases on the surface of the tip and sample. 3 T. Thundat, R. J. Warmack, D. E Allison, L. A. Bottomley, A. Lourenco, and T. L. Ferrell, J. Vac. Sci. Technol. A. 10, 630 (1992).

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Intermittent Contact Mode. Presently, most biological samples are imaged us-

ing intermittent contact. In this mode, the cantilever is oscillated at its natural resonance or driven at some selected frequency. Separation of the sample and tip is reduced until the oscillation of the cantilever is dampened. Images are acquired by tracking the shift in either amplitude or phase of oscillation as a function of the xy coordinates of the tip above the sample. Maps of sample topography are produced by recording shifts in amplitude whereas maps of sample compliance are produced by recording shifts in phase. Resolution is enhanced with intermittent contact imaging compared with contact imaging because the reduced tip wear allows for imaging with sharper and more delicate probe tips. Sample and tip damage is reduced because the tip is not shearing the surface; rather, its motion is always normal to the sample. Sticking of the tip to the surface is minimized because tip momentum is sufficient to overcome surface adhesion forces. The method by which the cantilever is oscillated varies among microscope manufacturers. Digital Instruments (Santa Barbara, CA) uses TappingMode whereas Molecular Imaging (Phoenix, AZ) uses MAG mode. In TappingMode, a piezoelectric oscillator is placed in contact with the cantilever holder and the entire cantilever assembly is oscillated at a desired frequency.4 The MAG mode oscillates a cantilever, coated with a magnetic material, with an induction coil placed beneath the sample. The oscillating magnetic field emanating from the coil drives the cantilever.5 Cantilever oscillation amplitudes are reduced compared with TappingMode. The relative advantages and limitations of each method are the subject of an ongoing debate in which we chose not to engage. In the context of this chapter, we shall treat both modes as equivalent. Tip and Scanner Calibration Cantilevers, tips, and the scanner are critical components of the microscope. Commercially available rectangular and V-shaped cantilevers are appropriate for nucleic acid imaging. The latter provides lower mechanical resistance to vertical deflection and high resistance to lateral torsion. 6 The size, shape, and material composition of an SFM tip have important consequences on the imaging of nucleic acids. Because the resultant scanning probe microscopy (SPM) image is always some convolution between the tip shape and image topography, tip artifacts will always be intrinsic to the technique.

4 D. A. Waiters, J. P. Cleveland, N. H. Thomson, P. K. Hansma, M. A. Wendman, G. Gurley, and V. Elings, Rev. Sci. lnstru. 67, 3583 (1996). 5 W. H. Han, S. M. Lindsay, and T. W. Jing, Appl. Phys. Lett. 69, 4111 (1996). 6 R. Howland and L. Benatar, in "A Practical Guide to Scanning Probe Microscopy." Park Scientific Instruments, Sunnyvale, California, 1996.

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Even though only the end of an SFM probe tip interacts with the molecule, the size of the tip dramatically affects the quality of images of nucleic acids. Tip irregularities away from the end are of importance only when imaging larger structures such as nucleosomes of chromatin. 7 For simplicity, the shape of the end of the tip is routinely assumed to be spherical when estimating actual feature widths from the apparent widths seen in micrographs. 8 The relationship between the observed diameter, Dobserved, and tip radius, Rtip, is given in Eq. (1). Dobserved =

4

-I/2 (Rtiprfeature) -

(1)

Equation (1) is applicable only when the radius of the feature, rfeature, is less than or equal to the radius of tip. When Rti p
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vertical quarters. Deformation of the tube occurs when a strain is induced in the cylinder through appropriately applied voltages. This strain causes the cylinder to bend, thus moving the sample (or tip) laterally. An electrode attached to the center of the tube provides for extension or contraction of the tube vertically, thereby moving the sample (or tip) up or down. Bending of the scanner tube in the horizontal direction causes a bowing artifact in large-scale images.15 Bowing occurs because as the scanner bends, the probe tip moves out of the plane of the sample. The scanner must then contract to maintain a contact cantilever deflection angle, resulting in an image that appears to be curved. SFM images of single nucleic acid molecules are often "flattened" through use of graphics software provided with the microscope, even when fiat substrates are used. Image resolution is also related to scanner performance, size of the scan area, and the specifications of the analog-to-digital converter (ADC) used for converting the analog photodiode detector signal to a digital signal. Lateral resolution is determined by the scan domain and the number of samples per scan line whereas vertical resolution is determined by the scanner's maximum z displacement, the fraction sampled by the ADC, and the number of bits of the ADC. For example, when a 1.0-#m 2 image is acquired with 512 samples per line and scan lines per image, the lateral resolution is 1.95 nm (distance tip traverses divided by the number of samples per line). If this image is acquired with a scanner with 6.0-#m vertical displacement sampled over this entire z range by a 16-bit ADC, the vertical resolution is 0.9 A (z displacement divided by number of digitization bits). Tip geometry impacts only the apparent feature width, not its height. Calibration of the scanner is required for accurate measurement of nucleic acid contour or persistence lengths. Because lead zirconium titanate piezoelectric materials exhibit intrinsic nonlinearity, hysteresis, creep, aging, and cross-coupling effects, frequent calibration of the scanner is recommended) 6 Calibration standards are readily available, reducing relative errors in length measurements of single molecules below 3%. Colloidal gold can be used as an internal standard when imaging biomolecules. Colloidal gold particles have multiple uses as three-dimensional, incompressible, monodisperse, and spherical SFM imaging standards as well as particles that can be exploited to characterize scanning tip geometry. 17.~s Biological molecules are "soft" and require lower forces for successful imaging. ~9,20The total force exerted by the tip is composed of the force resulting 1_5j. p. Starink and T. M. Jovin, Surf Sci. 359, 291 (1996). 16 R. Durselen, U. Grunewald, and W. Preuss, Scanning 17, 91 (1995). 17 j. Vesenka, S. Manne, R. Giberson, T. Marsh, and E. Henderson, Biophys. J. 65, 992 (1993). 18 j. W. Carlson, B. J. Godfrey, and S. G. Sligar, Langmuir 15, 3086 (1999). 19 T. E. Schaffer, J. P. Cleveland, E Ohnesorge, D. A. Waiters, and P. K. Hansma, J. Appl. Phys. 80, 3622 1996. 20 C. A. Putman, K. O. Vanderwerf, B. G. Degrooth, N. E Vanhulst, and J. Greve, Appl. Phys. Lett. 64, 2454 1994.

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from deflection of the cantilever, adhesive forces, and capillary forces. Typically, when imaging nucleic acids, forces less than 10 nN are required for high-quality micrographs. Capillary forces between water layers found on the tip and sample surfaces in ambient air exceed this value. These forces can be minimized by performing the experiment under low-humidity conditions or under fluid. Imaging Drug-DNA Complexes

Preparing Substrates For SFM imaging, substrates should be atomically flat, exhibit strong affinity for the molecule to be imaged, and have minimal attraction to the tip. Commonly used atomically flat substrates are mica, silicon, and thin films of gold evaporated onto a rigid support (mica, glass, or silicon). Mica is a two-dimensional material that, when cleaved along crystallographic planes, presents an atomically smooth surface over several hundred square microns. Silicon wafers, polished to subnanometer roughness levels, afford an even larger area for imaging plasmid DNA. Evaporated gold films are also used as substrates, but grain size markedly depends on the method of preparation. 21 Atomically flat domains of several hundred square microns are possible when the films are prepared by the template stripping method. 22 Selection of the substrate impacts the immobilization mode(s) available. Established protocols for immobilization of nucleic acids involve either electrostatic immobilization. 23,24 or covalent interaction25 28 between the molecule and the surface. Electrostatic immobilization is quick and requires no modifications to the nucleic acid. Covalent attachment pins the nucleic acid to the surface at known points on the molecule and affords a stronger attachment to the surface. Cleavage of mica leaves a surface that readily adsorbs divalent cations. The resulting surface attracts the negatively charged phosphate groups on nucleic acids. This strategy is applicable to imaging under buffer29-3j and in air. 23

21 j. A. DeRose, T. Thundat, L. A. Nagahara, and S. M. Lindsay, Surf Sci. 256, 102 (1991). 22 R Wagner, M. Hegner, H. J. Guntherodt, and G. Semenza, Langmuir 11, 3867 (1995). 23 j. Vesenka, M. Guthold, C. L. Tang, D. Keller, E. Delaine, and C. Bustamante, Ultramicroscopy 42-44, 1243 (1992). 24 y. L. Lyubchenko, A. A. Gall, L. S. Shlyakhtenko, R. E. Harrington, B. L. Jacobs, P. 1. Oden, and S. M. Lindsay, J. Biomol. Struct. Dyn. 10, 589 (1992). 25 A. J. Leavitt, L. A. Wenzler, J. M. Williamsand, and T. R Beebe, J. Phys. Chem. 98, 8742 0994). 26 C. Bamdad, Biophys. J. 75, 1997 (1998). 27 E Wagner, M. Hegner, E Kemen, E Zangg, and G. Semenza, Biophys. J. 70, 2052 (1996). 28 L. A. Chrisey, G. U. Lee, and C. E. O'Ferrall, Nucleic Acids Res. 24, 3031 (1996). 29 H. G. Hansma and D. E. Laney, Biophys. J. 70, 1933 (1996). 3o M. Fritz, M. Radmacher, J. R Cleveland, M. W. Allersma, R. J. Stewart, R. Gieselmann, E Janmey, C. E Schmidt, and E K. Hansma, Langmuir l l , 3529 (1995). 31 C. Bustamante, C. Rivetti, and D. J. Keller, Curt. Opin. Struct. Biol. 7, 709 (1997).

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Imaging under buffer requires adjustment of solution pH to maintain negatively charged DNA and careful choice of divalent cation. Hansma and co-workers 29,30 have shown Ni 2+, Co 2+, or Zn 2+ is needed to promote binding of DNA to mica surfaces for imaging under buffer. Individual molecules can be adsorbed and released by varying the concentration of these cations in the buffer. Imaging in air requires the presence of a divalent cation in the DNA loading solution and a means to remove unwarranted deposits of buffer salts. The latter is achieved either by copious rinsing of the DNA-laden mica surface or by using volatile buffers that will not leave a salt residue (e.g., ammonium acetate). 32 We typically use a loading sample containing DNA at a concentration between 0.1 and 10 #g/ml in 200 mM ammonium acetate, 5 mM MgC12, adjusted to pH 7.0 with NaOH, in 18-Mr2 water. A 10- to 50-/zl aliquot is incubated on the mica surface for 10 to 60 min, depending on the concentration of DNA and the desired number of molecules per square micrometer. After incubation the mica is dipped in water, a 1 : 1 (v/v) mixture of water and ethanol, and then anhydrous ethanol to wash away any excess salt deposits from the buffers. Excess liquid is "wicked" away with a Kimwipe and the disk is blown dry with clean compressed chlorofluorocarbon gas or dry N2 directed normal to the disk surface. The sample is stored in a desiccator until imaged under low-humidity conditions (dry He or N2 atmosphere). Electrostatic immobilization of DNA to other substrates requires that these surfaces be chemically modified. The most popular method uses 3-aminopropyltriethoxysilane (APTES) to create an electropositive layer on mica, silicon, or glass. 24 Vapor transfer is preferred over direct contact of APTES-containing solutions to minimize the formation of multilayers on the surface. An advantage of this method is that APTES-derivatized surfaces can be stored in the dry state for long periods of time. Loading of the DNA on this surface is achieved by the same protocol as described; however, buffers need not contain divalent cations. Nucleic acids can be covalently attached to gold surfaces. 25-2s DNA molecules with either thiolated phosphates 25 or pendant thiol groups 26 will spontaneously from Au-S bonds with the surface. Alternatively, gold, mica, silicon, or glass surfaces can be modified with heterobifunctional linkers reactive to DNA. Commonly used heterobifunctional linkers contain thiol or silane moieties at one end and succinimidyl, carboxylic acid, or maleimide moieties at the other end. 27'2s Thiolated linkers react with gold surfaces whereas silanated linkers react with oxide functionality on mica, silicon, and glass. Pendant amino groups on the DNA react with succinimidyl or carboxylic acid moieties [in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] forming peptide linkages. Pendant thiol groups react with maleimide moieties forming thioether linkages. DNA has also been linked to mica with a psoralen-terminated alkylsilane. 33 With this 32 E. Droz, M. Taborelli, T. N. Wells, and R Descounts, Biophys. J. 65, 1180 (1993). 33 L. S. Shlyakhtenko, A. A. Gall, J. J. Weimer, D. D. Hawn, and Y. L. Lyubchenko, Biophys. J. 77, 568 (1999).

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heterobifunctional linker, the DNA molecule is held on the surface by intercalation (via hydrogen bonding and Jr-stacking interactions). This approach suggests that site-specific attachment of DNA to surfaces may be possible with monolayers possessing functionality that binds within the grooves of double-stranded DNA (dsDNA). Assayforlntercalation. Intercalators are flat, polyaromatic molecules that bind to duplex DNA by insertion between the base pairs without breaking Watson-Crick hydrogen bonds. Each intercalator lengthens the DNA by an amount equivalent to the van der Waals thickness of the intercalating moiety and unwinds the double helix. Although groove-binding ligands may cause changes in the tertiary structure of the nucleic acid (e.g., bending), none are known to lengthen the molecule. SFM provides a straightforward means for determining lengths of nucleic acids with high precision. Coury and co-workers have established SFM as a direct, rapid, and unambiguous assay for mode of binding of conventional and nonclassic ligands to DNA. 34-36 The assay directly measures the lengths of individual DNA molecules before and after incubation with DNA-binding ligands. DNA lengthening on ligand binding provides direct evidence of intercalation. Implicit in this assay is the assumption that the length and conformation of immobilized nucleic acid molecules are commensurate with their length and conformation in solution. Convincing evidence in support of this assumption has been obtained by these and other workers. 34'35'37 Measured lengths of electrostatically immobilized, isolated DNA molecules are, within experimental error, equal to the expected length for B-DNA. Molecular lengths are correlated with ethanol concentration in the DNA loading solution. Increasing ethanol concentration results in shorter molecular lengths, consistent with the well-established ethanol-induced DNA conformational change. At ethanol concentrations greater than 30% (v/v), measured lengths are equal to the expected length for A-DNA. 35'37 Interestingly, once immobilized on the surface, molecular lengths are fixed and no significant changes are observed over the course of several days or after rinsing in ethanol. Contour lengths can be determined by cumulative addition of line segment lengths, drawn along the molecule with off-line analysis software. 38'39 As apparent

34j. E. Coury, L. McFail-lsom,L. D. Williams, and L. A. Bottomley,Proc. Natl. Acad. Sci. U.S.A. 93, 12283 (1996). 35j. E. Coury, J. R. Anderson, L. McFaiMsom, L. D. Williams, and L. A. Bottomley,J. Ant. Chem. Soc. 119, 3792 (1997). 36D. T. Breslin, J. E. Coury, J. R. Anderson, L. McFail-isom, L. D. Williams, L. A. Bottomley,and G. B. Schuster, J. Am. Chem. Soc. 119, 5043 (1997). 37y. Fang, T. S. Spisz, and J. H. Hoh, Nucleic Acids Res. 27, 1943 (1999). 38 y. Fang, T. S. Spisz, T. Wiltshire, N. P. D'Costa, I. N. Bankman, R. H. Reeves, and J. H. Hoh, AnaL Chem. 70, 2124 (1998). 39T. S. Spisz, Y. Fang, C. K. Seymour,R. H. Reeves, J. H. Hoh, and I. N. Bankman, Med. Biol. Eng. Comput. 36, 667 (1998).

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widths of the molecules increase, bends in the molecule become obscured and can no longer be accurately measured, thus contributing to greater deviation from molecule to molecule. Thus, DNA molecules with ambiguous topology should be excluded. The contribution of tip shape and diameter to the contour length measurement depends on the length of the strand. Tip diameter has a minimal impact on measurements of long DNA fragments (e.g., linearized plasmids). For example, when imaging a linearized plasmid containing 10,305 bp with a 20-nm tip radius, tip effects contribute only ",~18 nm (or 0.5%) of the measured DNA length. However, when the expected contour length of the DNA strand is equal to or less than the diameter of the tip, measured lengths must be corrected for tip effects. This is illustrated in Fig. 2. To identify an intercalator by using the Coury assay, 34 a series of solutions containing the ligand and DNA in varying concentration ratios are prepared,

FIG. 2. Topographic images of DNA on mica, illustrating the impact of tip shape on feature dimension. (a) Linearized pBBHb in B-DNA confimaation. Bar: 500 nm. The contour length of the molecule is 3500 nm. (b) Two short dsDNA molecules (20 bp each) joined by a disulfide linkage, that is, [5'-S-(CH2)6-GCGAT(A)IoACTGG-3']. Bar: Apparent length of 50 nm.

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, . - ~ .....

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300

400

500

Intercalator Concentration,

FIG. 3. Plot illustrating the impact of exclusion number on the length of a completely saturated intercalator-DNA complex. Lengths were computed on the basis of 10,300 bp for the dsDNA, 0.34 nm per binding event, and a binding affinityof 5.0 x 104. n = 2 (--), 3 (<>),4 (©), or 5 (0).

applied to atomically flat substrates, and imaged. Images of isolated molecules are obtained for each solution and at several locations on the substrate. Contour lengths are determined, collected, and plotted as a function of total ligand concentration. If the ligand intercalates, molecular lengths should increase with ligand concentration. The length at saturation provides a simple means to determine the exclusion number for the intercalator. If, at saturation, a ligand occupies every potential site (every dinucleotide step), the length of B-DNA is twice that of the unintercalated molecule. If, at saturation, a ligand occupies only every other dinucleotide step, the length of B-DNA is 50% longer than that of the unitercalated molecule. Thus, the exclusion number, n, can be computed from the saturation length, Lsat, and the measured length obtained in the absence of intercalator, L0, according to Eq. (2): n -- -

L0

-

Lsat --

L0

(2)

The impact of exclusion number in plots of measured DNA length versus ligand concentration is shown in Fig. 3. Noninteger values for the exclusion number are

[ 1 1]

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Intercalator Concentration, ixM FIG. 4. Plot illustrating the impact of binding affinity on the curvature of a length of DNA versus intercalator concentration. Lengths were computed on the basis of 10,300 bp for the dsDNA, 0.34 nm per binding event, and an exclusion number of 2. Binding affinity of 5.0 x 104 (--), 7.5 × 10a (O), 1.0 × 105 (O), 2.5 x 105 (O), and 5.0 × 105 (O).

possible with this method. Noninteger exclusion numbers indicate either sequencespecific binding 4° or multiple configurations existing along the DNA molecule. 41 Multiple configurations originate from out-of-plane substituents on the intercalator blocking entrance to other binding sites, adjacent or not. Random filling of binding sites creates gaps along the DNA that result in apparent noninteger exclusion numbers. The binding affinity of an intercalator can be determined from the rising portion of the measured DNA length-versus-ligand concentration plot. Figure 4 shows the impact of increasing intercalator binding affinity on the curvature of DNA lengthversus-intercalator concentration traces. Binding affinity of an intercalator, K is defined as [occupied intercalation sites] K = (3) ]unoccupied intercalation sites] [free drug] The fractional increase in DNA length at a given ligand concentration indicates the fraction of intercalation sites occupied. These concentrations can be explicitly 40 j. j. Correia and J. B. Chaires, Methods Enzymol. 240, 593 (1994). 41 j. D. McGhee and P. H. yon Hippel, J. MoL Biol. 86, 469 (1974).

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BIOPHYSICALAPPROACHES

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related to measured lengths by the expressions [Occupied intercalation sites] = ( ~ - ~ - ) occ pie interca a ion

[DNA]

(4)

=

[Freedrug]=Cf=llo-(LaL'~))[DNA]]

(6)

where L is the length of intercalated DNA molecule, Cf is the free drug concentration, I0 is total intercalator concentration, B is the number of base pairs per DNA molecule, and a is the lengthening per intercalation event. Direct substitution of these equations into Eq. (3) results in a function that is transcendental with respect to L. Solving this equation for I0 yields Eq. (4), which can be used to extract binding parameters from SFM length data. I°:K[B_

( g _ ~ ) ] +[DNA]

T

(7)

L0, [DNA], and B are, in most circumstances, precisely known experimental constants. Nonlinear least-squares analysis may be applied to compute values of K, n, and a from experimental data. Alternatively, the number of parameters to be computed may be reduced by setting a = 3.4 A, the van der Waals thickness of most intercalators. Whenever conformational changes in DNA are possible, caution should be exercised in assigning this variable as a constant. Note that of the parameters n and a are strongly correlated, using nonlinear least-squares fitting routines. Binding affinities determined in this way are comparable to values determined by conventional assays. SFM length data can be satisfactorily fit by Scatchard models 42 as well as more sophisticated models incorporating cooperativity, size exclusionf and binding heterogeneity.43 The Scatchard model is 1" --

Cf

:

(8)

K ( N - r )

where r is the number of ligands bound per base pair and N is the number of binding sites available per base pair (i.e., 1/exclusion number = The number of ligands per base pair can be computed from microscopic measurements according to Eq. (8):

1/n).

r

--

L -L0

aB

(9)

42 G. Scatchard, Ann. N.Y. Acad. Sci. 51, 660 (1949). 43 j. B. Chaires, in "Advances in DNA Sequences Specific Agents," (L. H. Hurley, ed.), p. 3. JAI Press, Greenwich, Connecticut, 1992.

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Empirical values of K and n are obtained by nonlinear least-squares fitting of the model to data. McGhee and von Hippel have put forth more complicated models incorporating cooperative interactions between ligands. 41 Implicit in either approach is the assumption that the independent variable r is precisely known. This assumption may not be valid when r is computed from SFM lengthening data. Uncertainties in length measurements propagate in computed values of r and Cf; this is especially significant at low ligand loading levels. Site oflntercalation. Coury and co-workers 44 have also demonstrated the utility of SFM for determining the site of intercalation. Biotinylated psoralen was intercalated into a linearized plasmid and the binding site was marked by reaction of the biotin moiety with streptavidin bound to a colloidal gold bead. Streptavidin alone can be used because the biotin-streptavidin conjugate is readily visualized in SFM images of DNA. Alternative labeling strategies now available include reaction of pendant amino or thiol termini on the intercalator with C60 or gold nanoclusters possessing succinimidyl or thiol surface functionality. 45 Assay f o r Groove Binding

Parts of each base pair are exposed in the two distinct grooves of doublehelical DNA. Thus, drugs that bind in either the major or minor groove do so with a high degree of sequence specificity. As yet, no SFM-based assay has been developed that provides a direct measure of ligand binding in the major or minor groove. Commercially available SFM tips are presently too broad to provide direct visualize the major or minor groove of the double helix. Such images require tips with diameters approximating the dimensions of the grooves. Until angstrom-sized tips become available, visualization of groove-bound ligands is limited to either changes in DNA strand height (contrast) and width, or to labeled ligands. Hansma et al. have observed differences in curvature between uncomplexed DNA and DNA bound with distamycin, a well-known minor groove binder. 46 To our knowledge, this is the only published study involving minor groove binding. Binding in the minor groove can be assessed by a competitive binding approach, 34-36 For example, ethidium will compete with a drug that binds exclusively in the minor groove. On intercalation, the ethyl and phenyl substituents of ethidium are resident in and block the DNA minor groove, whereas the major groove remains sterically unencumbered. Thus, when the ratio of these competing ligands is systematically varied, the length of the DNA complex should scale with ethidium concentration but be significantly less than that observed in the absence of the minor groove-binding ligand. Note that samples must be prepared and imaged 44j. E. Coury,L. McFail-lsom,S. Presnell, L. D. Williams,and L. A. Bottomley,,I. Vac. Sci. Technol. A 13, 1746(1995). 4.5K. Sugawara,S. Tanaka,and H. Nakamura,Anal. Chem. 67, 299 (1995). 46 H. G. Hansma,K. A. Browne, M. Bezailla,and T. C. Bruice. Biochemisow 33, 8436 (1994).

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in the same manner employed for intercalator-DNA complexes alone. The major groove-binding mode can be identified through competitive binding experiments as well. Ditercalinium, an intercalator with substituents that obstruct the major but not the minor groove, competes with major groove binders. Nogalamycin, an intercalator with substituents in both grooves, will compete with all groove-binding ligands. Several workers have acquired images of oligonucleotides bound in the major groove.4%49 Hansma et al. observed triple-stranded DNA structures in images of poly(dA) • poly(dT) and poly(dC), poly(dG).47 These structures were twice as high as double-stranded DNA and the same width. Cherny and co-workers4s presented SFM images of a G,A-containing triplex-forming oligomer conjugated to a long plasmid DNA. Triplex regions were 40.4 nm higher than the corresponding duplex regions. Pfannschmidt and co-workers49 carried out site-specific labeling of closed circular DNA by using triplex-forming oligonucleotides containing a reactive psoralen at the 5' end and a biotin at the 3' end. The probes were directed to different target sites on plasmid DNA and photocross-linked to the target to increase stability. The covalent adduct DNAs were visualized by SFM, using avidin or streptavidin as protein tags for the biotin group on the oligonucleotide probes. Height measurements in the region of the triplex were consistent with those observed by Hansma et al. and by Chemy et al. Imaging Protein-DNA Complexes Analysis of the structure of protein-nucleic acid complexes is an important area of application of SFM. Protein-DNA complexes are prepared and immobilized electrostatically onto mica substrates by the protocol described above for immobilizing drug-DNA complexes. Structural details of the protein-nucleic acid complex are obtained from analysis of images acquired in air or under buffer. For example, SFM images acquired by Cary et al. 5° revealed sequence-independent DNA looping by Ku protein and the DNA-dependent protein kinase, two proteins responsible for DNA repair and genetic regulation. Images by Pang et al. 51 showed that Ku protein binds predominantly to the ends of double-stranded linear DNA, does not bind to circular plasmids, and joins two ends of DNA together. These observations suggest that Ku protein may play a role in physically orienting DNA 47 H. G. Hansma, I. Revenko, K. Kim, and D. E. Laney, Nucleic Acids Res. 24, 713 (1996). 48 D. ][. Cherny, A. Fourcade, E Svinarchuk, R E. Nielsen, C. Malvy, and E. Delain, Biophys. J. 74, 1015 (1998), 49 C. Pfannschrnidt, A. Schaper, G. Heim, T. M. Jovin, and J. Langowski, Nucleic' Acids Res. 24, 1702 (1996). 5o R. B. Cary, S. R. Peterson, J. Wang, D. G. Bear, E. M. Bradbury, and D. J. Chen, Proc. Natl. Sci. U.S.A. 94, 4267 (1997). 51 D. Pang, S. Yoo, W. S. Dynan, M. Jung, and A. Dritschilo, Cancer Res. 57, 1412 (1997).

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for ligation by binding the ends of adjacent DNA molecules. Valle et al. 52 studied the interaction of DNA with bacteriophage 429 connector. SFM images revealed that the circular DNA-protein complex bends the strands to a mean angle of 132 ° and that the DNA binds to the outer side of the protein. Garcia and co-workers 53 determined that M . H h a I , a cytosine C5 DNA methyltransferase, causes only a 2 ° bend on binding its recognition site. In contrast, images of the M . E c o R I , an adenine N6 DNA methyltransferase, shows an average bend angle of ~ 5 2 °. This distortion of DNA conformation by M . E c o R I is important for sequence-specific binding. Formation of protein-DNA complexes can also be used to mark specific sites on the nucleic acid. For example, Allison and co-workers 54'55 used mutant E c o R I endonuclease binding to specific nucleotide sequences to create distinctive features in images of plasmids. Binding of the mutant endonuclease to specific nucleotide sequences produced distinctive features in images of plasmids. By measuring contour length distances between these features, physical maps of individual cosmid DNAs were produced by direct SFM imaging with an accuracy of better than 1%. This method is faster and more accurate when compared with conventional electrophoretic mapping methods. Klinov and co-workers 56 performed high-resolution mapping of individual plasmids and cosmids, using RNA probes specific for long terminal repeats within these DNA. The RNA probes formed so-called R-loops that, when stabilized by glyoxal, were readily imaged after chemisorption of the conjugate onto Mg2+-modified mica. R-loop positions were accurate to 0.5% of the cosmid length. SFM imaging of protein-DNA complexes has been used to gain insight into their function. For example, the repression of transcription of two overlapping promoters of the g a l operon in E s c h e r i c h i a coli requires Gal repressor (GalR) and the histone-like protein HU. Lyubchenko et al. 57 imaged Gal-DNA complexes with proteins and found that GalR mediated DNA looping in which HU plays an obligatory role by helping GalR tetramerization. Nettikadan and co-workers 58 investigated transcription factor AP2 binding to DNA. SFM images proved that protein-binding sites can be mapped over a few kilobases of target DNA and 52 M. Valle, J. M. Valpuesta, J. L. Carrascosa, J. Tamayo,and R. J. Garcia, J. Struct, BioL 116, 390 (1996). 53 R. A. Garcia, C. J. Bustamante, and N. O. Reich, Proc. Natl. Acad. Sci. U.S.A. 93, 7618 (1996). 54 D. P. Allison, P. S. Kerper, M. J. Doktycz,T. Thundat, P. Modrich, F. W. Larimer, D. K. Johnson, P. R. Hoyt, M. L. Mucenski, and R. J. Warmack, Genomics 41,379 (1997). 55p. R. Hoyt, M. J. Doktycz,P. Modrich, R. J. Warmack,and D. P. Allison, Ultramicroscopy 82, 237 (2000). 56D. V. Klinov, I. V. Lagutina, V. V. Prokhorov,T. Neretina, P. P. Khil, Y. B. Lebedev, D. I. Cherny, V. V. Demin, and E. D. Sverdlov,Nucleic Acids Res. 26, 4603 (1998). 57y. L. Lyubchenko,L. S. Shlyakhtenko,T. Aki, and S. Adhya,Nucleic Acids Res. 25, 873 (1997). 58 S. Nettikadan, F. Tokumasu, and K. Takeyasu,Biochem. Biophys. Res. Commun. 226, 645 (1996).

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that multimerization state of DNA-binding proteins can be determined simply by measuring the sizes of proteins bound of the DNA. Rippe et al. 59 used SFM to study transcriptional activation of E. coli RNA polymerase ¢r54 at the glnA promoter by the constitutive mutant of the nitrogen regulatory protein C. DNA-protein complexes were deposited on mica and imaged in air. By choosing appropriate conditions, the structure of various intermediates in the transcription process could be visualized and analyzed. Smith et al. 6° investigated the complexes formed between purified poly(A)-binding protein and poly(A) RNA, using SFM. Poly(A)-binding protein is an RNA-binding protein that binds specifically to the poly(A) tail of mRNAs in eukaryotes. SFM images revealed that the protein formed variable-size complexes bound lengthwise along the RNA. Poly(A) RNA alone appeared to contain a knoblike structure that largely disappeared once the protein was bound. Margeat and colleagues 61 visualized the protein-protein and protein-DNA complexes involved in transcriptional regulation by the trp repressor (TR). Plasmid fragments bearing the natural operators trpEDCBA and trpR, as well as nonspecific fragments, were deposited onto mica in the presence of varying concentrations of TR and imaged. Specific and nonspecific complexes of TR with DNA are found, as well as free TR assemblies directly deposited onto the mica surface, Their findings suggest protein-protein interactions serve a role in transcriptional regulation by the trp repressor. Perhaps the most exciting application of SFM lies in the real-time visualization of protein-DNA complexation. The major challenge is to find imaging conditions that promote adsorption of protein-DNA complexes onto atomically fiat substrates without destabilizing or inhibiting movement of the complex. Once these conditions have been found, reconstruction of images acquired over time provides a straightforward means for analyzing motion in biological systems. Thomson and co-workers 62 prepared recombinant RNA polymerase containing histidine tags (His-RNAP) on the C terminus and immobilized them onto ultraflat gold via a mixed monolayer of alkanethiols. Specific binding of this molecule to a 42-base circular single-stranded D N A template was confirmed by in situ SFM images showing the production of huge RNA transcripts. Bustamante and co-workers 63'64 obtained tapping-mode SFM images that demonstrated the diffusion of E. coli RNA polymerase along DNA. Direct evidence 59K. Rippe, M. Guthold,E H. von Hippel, and C. J. Bustamante,Mol. Biol. 270, 125 (1997). 60B. L. Smith,D. R. Gallie, H. Le, and P. K. Hansma,J. Struct. Biol. 119, 109 (1997). 61E. Margeat,C. Le Grimellec,and C. A. Royer,Biophys. J. 75, 2712 (1998). 62N. H. Thomson,B. L. Smith, N. Almqvist,L. Schmitt, M. Kashlev,E. T. Kool,and P. K. Hansma, Biophys. J, 76, 1024 (1999), 63E. T. Kool, P. K. Hansma, M. Kashlev, S. Kasas, N. H. Thomson, B. L. Smith, H. G. Hansma, X. Zhu, M. Guthold, and C. Bustamante,Biochemistry 36, 461 (1997). 64C. Bustamante,M. Guthold, X. Zhu, and G. Yang,J. Biol. Chem. 274, 16665 (1999).

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of facilitated targeting of RNA polymerase by intersegment transfer and possibly hopping (intradomain association and dissociation) was obtained for the first time. Ternary intermediates, in which RNA polymerase appears to be simultaneously bound to two DNA segments, were directly observed during intersegment transfer events. In some image sets, transfer events were preceded and followed by sliding processes. Their results provide additional insight into the mechanism by which RNA polmerase searches for the promoter, a key step in transcription. Their highresolution images and analysis provided the first direct evidence of individual complexes involved in transcription. Summary SFM is a viable and effective method for determining the mode of binding, the extent of binding, and the site of binding of intercalators to nucleic acids. Establishing the presence of a groove-bound ligand can be achieved either by competitive binding experiments with a well-defined intercalator (minor groove) or by changes in apparent contrast {major groove). In our opinion, SFM has an important role in resolving the structural polymorphisms for small molecule-DNA complexes. Application of these assays in the study of polyintercalator molecules is currently underway in our laboratory. SFM is an important, new tool in the study of protein-DNA complexes. New insights into the structure and function of these complexes are enabled by real-time visualization. Currently the temporal resolution of the SPM limits the degree to which definitive rate data can be determined. Several binding and unbinding events could take place in the time it takes to acquire one image. New developments in SFM technology will allow faster scanning and will improve the temporal resolution of so-called SFM movies. To this end, the Hansma group is developing small cantilevers 65 and improved optical deflection systems 66 to enable intermittent imaging at scanning rates of 1.7 sec per image. These improvements will enable SFM visualization of complex biological processes as they occur, one molecule at a time. Acknowledgments Financial supportfrom the ONR-sponsoredGeorgiaTech MolecularDesign Institute is gratefully acknowledged.We thankthe followingindividualsfor their insights, ingenuity,and involvementin the developmentof SFM assaysfor drug-DNAbinding: JosephCoury,JaimieAndersen,LorenWilliams, Lori McFail-Isom,and Brad Chaires. 65M. B. Viani, T. E. Schaffer, G. T, Paloczi, L. I. Pietrasanta, B. L. Smith, J. B. Thompson, M. Richter, M. Rief, H. E. Gaub,K. W. PLaxco,A. N. Cleland, H. G. Hansma,and P. K. Hansma,Rev. Sci. lnstr. 70, 4300 (1999). 66T. E. Schafferand P. K. Hansma,J. Appl. Phys. 84, 4661 (1998).