Langmuir 2002, 18, 5333-5336
5333
Using Force Spectroscopy To Investigate the Binding of Complementary DNA in the Presence of Intercalating Agents Mark P. Schotanus, Kimberly S. Aumann, and Kumar Sinniah* Department of Chemistry & Biochemistry, Calvin College, 3201 Burton Street SE, Grand Rapids, Michigan 49546 Received February 27, 2002. In Final Form: May 16, 2002 Small molecules are known to bind to DNA via simple intercalation, which leads to the unwinding of the DNA helix. We have demonstrated using force spectroscopy a significant reduction in the unbinding forces measured in a 16-mer DNA duplex in the presence of sequence specific and nonspecific intercalating agents, 3,6-diaminoacridine and ethidium bromide. We also explore the effect of varying the intercalator strength on the unbinding forces measured from the DNA duplexes. At very dilute concentrations of the intercalating agents, the unbinding forces measured are similar to those measured in a hybridizing buffer.
Introduction 1
A number of chemotherapeutic drugs bind to DNA. Since such drug molecules have pharmacological and medical importance, there is intense interest in understanding the mechanism of the drug-DNA interactions. Furthermore, the drug-DNA complexes are known to adopt as yet unresolved structural conformations.2 Small molecules are known to bind to DNA via simple intercalation, where the aromatic ring of the intercalator slips between the stacked base pairs of duplex DNA.3 More complex intercalators have substituents that are positioned in the minor groove, leading to both specific and nonspecific interactions.4 A number of methods, including the measurement of DNA melting curves5 and optical techniques such as absorbance, fluorescence, circular dichroism,6 and reflectometric interference,7 have been used to study the interactions of intercalating agents with DNA. In this paper we report the use of force spectroscopy to investigate directly the effect of intercalatants on DNA duplexes. This method is not limited by the size or optical properties of the ligand and has the distinct advantage of directly probing the intermolecular interactions. Force spectroscopy has been a useful technique to measure molecular interactions between the probe tip and samples immobilized with chemical or biological molecules. Although early experiments showed a clear discrimination between adhesion forces arising from * Corresponding author. Phone: 616-957-6058. Fax: 616-9576501. E-mail:
[email protected]. (1) (a) Bailey, C. Curr. Med. Chem. 2000, 7, 39. (b) Yang, X. L.; Wang, A. H. Pharmacol. Ther. 1999, 83, 181. (c) Murray, V. Prog. Nucleic Acid Res. Mol. Biol. 1999, 63, 367. (d) Hortoba´gyi, G. N. Drugs 1997, 54, 1. (e) Chen, H.; Liu, X.; Patel, D. J. J. Mol. Biol. 1996, 258, 457. (f) Baguley, B. C. Anticancer Drug Des. 1991, 6, 1. (g) Denny, W. A. Anticancer Drug Des. 1989, 4, 241. (h) Brown, J.; Imam, H. Prog. Med. Chem. 1984, 21, 170. (2) Herzyk, P.; Neidle, S.; Goodfellow, J. M. J. Biomol. Struct. Dyn. 1992, 10, 97. (3) Lerman, L. S. Proc. Natl. Acad. Sci. U.S.A. 1963, 49, 94. (4) (a) Chaires, J. B. Biopolymers 1997, 44, 201. (b) Jain, S. C.; Sobell, H. M. J. Biomol. Struct. Dyn. 1984, 1, 1161. (5) Mahler, H.; Goutarel, R.; Khuong-Huu, Q.; Ho, M. Nucleic Acid. Int. 1966, 5, 2177. (6) Wang, A. In Interactions between antitumor drugs and DNA, Nucleic Acids and Molecular Biology; Eckstein, F., Lilley, D., Eds.; Springer-Verlag: New York, 1987; Vol. 1. (7) Piehler, J.; Brecht, A.; Gauglitz, G.; Zerling, M.; Maul, C.; Thiericke, R.; Grabley, S. Anal. Biochem. 1997, 249, 94.
different functional groups attached to probes and surfaces,8 the interpretation of the results had to take into account effects arising from solvent exclusion, tip geometry, and coverage of molecules on the tip and surface. More recently, Evans9 has shown that varying loading rates by several orders of magnitude in force spectroscopy measurements will lead to the rupture of all noncovalent bonds, suggesting the magnitude of the forces measured in a typical force spectroscopy experiment is dependent on the loading rates applied. Hence, by varying the loading rates, an energy map of the binding potentials of single molecule interactions can be elucidated. However, keeping fixed pulling rates or a constant force in adhesion force measurements is nevertheless useful, as we have shown in this paper and from previous reports,8 but represents only a single point in the dynamic force spectroscopy measurement.10 In this study, the loading rates were not varied and were kept constant in order to make direct comparisons from the effect of DNA binding intercalators. Force measurements between complementary DNA duplexes have been studied by several groups with different DNA sequences and surface preparations.11 Typically, the unbinding force of complementary DNA strands is measured by attaching single-stranded DNA on the tip and the surface and then bringing the tip and the surface into contact for hybridization and retracting the piezo to unbind the DNA duplex formed. The resulting force-distance curve provides a measure of the unbinding force. A large number of the force curves show repulsive (8) (a) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. Science 1994, 265, 2071. (b) Green, J. B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys. Chem. 1995, 99, 10960. (c) Sinniah, S. K.; Steel, A. B.; Miller, C. J.; Reutt-Robey, J. E. J. Am. Chem. Soc. 1996, 118, 8925. (d) Noy, A.; Vezenov, D. V.; Lieber, C. M. Annu. Rev. Mater. Sci. 1997, 27, 381. (9) (a) Evans, E. Annu. Rev. Biophys. Biomol. Struct. 2001, 30, 105. (b) Evans, E. Faraday Discuss. 1998, 111, 1. (10) Hugel, T.; Seitz, M. Macromol. Rapid Commun. 2001, 22, 989. (11) (a) Lee, G. U.; Chrisey, L. A.; Colton, R. J. Science, 1994, 266, 771. (b) Boland, T.; Ratner, B. D. Proc. Natl. Acad. Sci. 1995, 92, 5297. (c) Noy, A.; Vezenov, D. V.; Kayyem, J. F.; Meade, T. J.; Lieber, C. M. Chem. Biol. 1997, 4, 519. (d) Mazzola, L. T.; Frank, C. W.; Fodor, S. P. A.; Mosher, C.; Lartius, R.; Henderson, E. Biophys, J. 1999, 76, 2922. (e) Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346. (f) Strunz, T.; Oroszlan, K.; Schafer, R.; Guntherodt, H. J. Proc. Natl. Acad. Sci. 1999, 96, 11277. (g) MacKerell, A. D.; Lee, G. U. Eur. Biophys. J. 1999, 28, 415. (h) Pope, L. H.; Davies, M. C.; Laughton, C. A.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Eur. Biophys. J. 2001, 30, 53.
10.1021/la025669t CCC: $22.00 © 2002 American Chemical Society Published on Web 06/07/2002
5334
Langmuir, Vol. 18, No. 14, 2002
electrostatic interactions with zero adhesion while a significant fraction of force curves show pull-off events that can be described as arising from nonspecific and specific interactions. Another approach to measuring the DNA duplex strength has been to stretch double-stranded DNA between the surface and a tip and to map out the structural transitions occurring upon stretching.12 This approach to studying DNA mechanics was used recently to investigate the binding of small molecules to long strands of DNA (3500-6260 base pairs) and compare changes in structural transitions.13 The unbinding forces measured from short stranded DNA duplexes differ markedly between force spectroscopy experiments carried out under fixed loading rate versus variable loading rate conditions. As a result, in fixed loading rate force spectroscopy experiments, the DNA unbinding forces for 12-14-mer DNA duplexes were found to be around 415-460 pN,11a,c,g while, in experiments in which the loading rates were varied, the rupture forces for short strands appear to be between 20 and 50 pN.11f,h These differences appear to depend on the contact forces applied between the tip and the surface during approach.11h In this paper, we report the use of fixed loading rate force spectroscopy to study the binding of intercalating agents to a 16-mer DNA duplex. These measurements were performed on complementary DNA strands in the presence of intercalating compounds, 3,6-diaminoacridine (proflavine) and ethidium bromide, as well as in a nonhybridizing buffer and in a hybridizing buffer. Experimental Section Materials. The DNA oligonucleotide 5′-(ACTG)4-3′ and its complementary sequence 5′-(CAGT)4-3′ were purchased from Research Genetics (Huntsville, AL). They were synthesized with a 6-mercaptohexyl linker on the 5′ end to attach to gold surfaces. The nucleic acid sequences were repeated non-self-complementary four-base units of ACTG and CAGT. Two buffers were employed for the force spectroscopy measurements and for the dilution of intercalating agents. 1.0 M phosphate buffer (pH 7) was prepared in deionized water (18.2 MΩ‚cm resistivity) obtained from a Barnstead water purifier (Fisher Scientific). A commercial buffer, sodium saline phosphate EDTA (SSPE) (Sigma-Aldrich, St. Louis, MO), was purchased at a concentration of 20× and diluted to 1× (10 mM phosphate at pH 7.4, 149 mM NaCl, 1 mM EDTA) using deionized water for hybridizing experiments. The intercalating compounds, 3,6diaminoacridine and ethidium bromide (Sigma-Aldrich, St. Louis, MO) were diluted in the SSPE buffer and made up to the appropriate concentrations. Substrate Preparation. Gold substrates were prepared by sputter deposition of a 20 nm Cr adhesion layer followed by 200 nm of gold (99.99% purity) evaporation onto glass microscopic slides in a vacuum evaporator (Denton Vacuum, NJ). During evaporation the base pressure inside the vacuum chamber was around 8 × 10-6 Torr. Prior to DNA immobilization, the gold substrates were cleaned in NoChromix (Godax Laboratories, Takoma Park, MD) for a minute and then dipped in concentrated nitric acid for another minute and washed thoroughly with water and ethanol. Cleaned gold surfaces were then incubated in a 0.5 µM thiolated single-strand DNA solution overnight prior to usage. Commercial Si3N4 cantilevers were prepared similarly with a 3:30 nm ratio of Cr/Au and placed immediately in a 0.5 µM thiolated single-strand DNA solution. Both tips and samples were incubated overnight prior to using them for force measurements. AFM images of the DNA from our lab (data not shown) and others14 show that single-stranded DNA forms a compact (12) (a) Rief, M.; Clausen-Schaumann, H.; Gaub, H. E. Nat. Struct. Biol. 1999, 6, 346. (b) Clausen-Schaumann, H.; Seitz, M.; Krautbauer, R.; Gaub, H. E. Curr. Opin. Chem. Biol. 2000, 4, 524. (13) Krautbauer, R.; Pope, L. H.; Schrader, T. E.; Allen, S.; Gaub, H. E. FEBS Lett. 2002, 510, 154.
Letters self-assembled monolayer when incubated overnight or longer in solution, with the bulk of the DNA lying closer to the surface, while a random distribution of DNA strands extends away from the surface, appearing as bright white spots on the background surface.14c Instrumentation. All experiments were carried out in a Nanoscope IIIa AFM (Digital Instruments) instrument equipped with a 14.5-µm scanner (vertical engagement E scanner). Force constants of DNA immobilized gold cantilevers were measured using the method described by Sader et al.15 and were found to be in the range between 0.032 and 0.036 N/m for the cantilevers used in these experiments. Force Spectroscopy Measurements. Force spectroscopy measurements were performed in a glass fluid cell (Digital Instruments) at 25 °C. Force measurements were carried out in solutions of nonhybridizing buffer (1.0 M phosphate, pH 7.0) and hybridizing buffer (SSPE, 10 mM phosphate at pH 7.4, 149 mM NaCl, 1 mM EDTA), with varying concentrations of 3,6-diaminoacridine (DAA) (1.5 mM, 50 µM, 15 µM) and ethidium bromide (EB) (1.27 µM, 12.7 nM, 12.7 pM). A fixed pulling rate of 2700 nm/s was used in all of our data collections. Data were collected over a time period of 2 h, and a total of 500-2000 force curves were analyzed for at least three probe-surface pairs per experiment in the specific buffer and/or intercalator solution. Although a recent study separated the unbinding force curves as occurring from specific and nonspecific events,11h no such attempt was made to separate force curves on the basis of the force-distance curve shape to avoid introducing a bias to the data analysis. All force-distance curves were analyzed by an automated method to remove the subjective influence of an operator by using the commercial software Scanning Probe Image Processor (Image Metrology, Denmark).
Results and Discussion The force-curve data obtained from force spectroscopy measurements for the complementary pair of strands were quantified; representative examples of the distribution of the adhesion forces for 1.0 M phosphate, SSPE, 1.5 mM 3,6-diaminoacridine, and 50 µM 3,6-diaminoacridine are shown in Figure 1; and a complete analysis of the data is shown in Table 1. As expected, the data indicate that 1.0 M phosphate does not provide the appropriate conditions for DNA hybridizing. The average adhesion force observed for the large number of events, 0.11 ( 0.06 nN, is most likely arising from nonspecific interactions, as has been observed previously on DNA duplex interactions by Noy et al.11c Since the single-stranded DNA on the tip and surface are negatively charged, effective screening of this charge is necessary for promoting hybridization. This screening is typically achieved by the addition of salts to the buffer. Although most DNA annealing experiments by force spectroscopy have been conducted at physiologic salt concentrations, it appears that differences in salt concentration have an effect on the unbinding force.11a,g In the presence of the hybridizing buffer (SSPE), we observe five distinct regions where adhesion forces arising from specific interaction are centered at 1.13 ( 0.09 nN (14% of the total force), 0.85 ( 0.09 nN (22%), 0.60 ( 0.09 nN (30%), and 0.32 ( 0.08 nN (25%), while the nonspecific interactions are centered at 0.06 ( 0.05 nN (9%) (cf. Figure 1b). We interpret that the 0.6 nN value corresponds to unbinding forces arising from the full-duplex of the 16mer, while the unbinding force measured at 1.13 nN is arising from the binding of two duplexes. The latter force (14) (a) Sam, M.; Boon, E. M.; Barton, J. K.; Hill, M. G.; Spain, E. S. Langmuir 2001, 17, 5727. (b) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; McPherson, L. D.; Potter, A. B.; Spain, E. M.; Allen, M. J.; Hill, M. G. Langmuir 1998, 14, 6781. (c) Huang, E.; Satjapipat, M.; Han, S.; Zhou, F. Langmuir 2001, 17, 1215. (15) Sader, J. E.; Larson, L.; Mulvaney, P.; White, L. R. Rev. Sci. Instrum. 1995, 66, 3789.
Letters
Langmuir, Vol. 18, No. 14, 2002 5335 Table 1. Unbinding Forces Measured in a 16-mer DNA Duplex with and without Intercalating Agents specific interaction, nN
intercalating agent
nonspecific force (nN)
1.0 M phospate (control) SSPE (control) 1.5 mM 3,6-diaminoacridine 50 µM 3,6-diaminoacrdine 15 µM 3,6-diaminoacridine
0.10 ( 0.04 (88%) 0.06 ( 0.05 (9%)
1.27 µM ethidium bromide 12.7 nM ethidium bromide
0.18 ( 0.08 (60%) 0.11 ( 0.05 (75%)
12.7 pM ethidium bromide
0.16 ( 0.04 (5%)
0.16 ( 0.05 (31%) 0.11 ( 0.06 (51%)
partial overlap
single duplex
0.34 ( 0.08 (12%) 0.32 ( 0.08 (25%) 0.60 ( 0.09 (30%) 0.32 ( 0.14 (100%) 0.65 ( 0.15 (12%) 0.34 ( 0.06 (16%) 0.65 ( 0.06 (11%) 0.50 ( 0.05 (20%) 0.38 ( 0.12 (38%) 0.25 ( 0.07 (20%) 0.42 ( 0.08 (5%) 0.30 ( 0.09 (64%) 0.60 ( 0.06 (17%) 0.48 ( 0.05 (12%)
multiple partial overlap
double duplex
0.85 ( 0.09 (22%)
1.13 ( 0.09 (14%) 1.09 ( 0.17 (44%) 1.32 ( 0.07 (2%)
a The percentages reported in parentheses refer to the number of pull-off events representing the fitted Gaussian distribution. The uncertainties reported are the widths of the Gaussians. All intercalating agents were used in 1× SSPE buffer.
Figure 1. Histogram of the unbinding forces measured between complementary DNA oligonucleotides between tips and surfaces in (a) 1.0 M phosphate buffer (pH 7.0), (b) SSPE (10 mM phosphate at pH 7.4, 149 mM NaCl, 1 mM EDTA), (c) 1.5 mM 3,6-diaminoacridine (DAA) in SSPE, and (d) 50 µM 3,6-diaminoacridine in SSPE. Solid lines are nonlinear fits to a Gaussian distribution, while dashed lines represent the Gaussians used to generate the solid lines.
is nearly twice the value measured from the unbinding of the full complement within experimental error. The adhesion force at 0.85 nN, which falls between the unbinding forces measured for a single and a double duplex, suggests the result is more likely from the partial binding of more than one duplex. The unbinding force measured at 0.32 nN is nearly half the force measured from the unbinding of a full-duplex, suggesting that it results from a partial binding of a single duplex. This interpretation of our results for the DNA sequence we chose agrees well with a similar DNA sequence used by Lee et al.11a The repeat sequence we chose should provide complementary overlaps of 16, 12, 8, or 4 base pairs, while
the latter two interactions are unlikely and were not observed in previous experiments as well.11a,g Lee et al. have shown that the unbinding force of a 16-mer duplex is 0.56 nN in 0.1 M NaCl (after correction),11a while it is 0.33 nN in 0.01 M NaCl.11g Our experimental value of 0.60 nN for the unbinding of a 16-mer duplex agrees well with the value reported by Lee et al.11a at a similar salt concentration. Although it is conceivable that using a nondilute tip may lead to a large number of interactions between the DNA on the tip and surface, the compact DNA layer formed with several individual DNA strands extending away from the surface on both the tip and the surface would result most likely in only a very few strands forming duplexes at the point of contact between the tip and surface. In the presence of the intercalator 3,6-diaminoacridine (1.5 mM), we observe a broad distribution of forces with an average adhesion force centered at 0.32 nN. This value is similar to the result obtained from partial overlap between complementary DNA in the hybridizing buffer, suggesting a clear weakening of the DNA duplex strength. This significant decrease in the unbinding force is consistent with the fact that the DNA duplex has to undergo conformational change in order for the 3,6diaminoacridine to intercalate. It has been shown that the dominant binding effect of 3,6-diaminoacridine in DNA is to intercalate between GC-GC base pairs or GC-AT base pairs;16 hence, the reduction in the adhesion force is most likely due to a lack of cooperativity in the duplex strand. It should be emphasized that this binding is reversible, since we were able to wash both the surface and the probe with a diluted SSPE solution and then measure adhesion forces in the SSPE buffer and obtain results consistent with Figure 1b. When the unbinding forces between the complementary DNA strands were measured in the presence of a 30fold dilute concentration of the intercalator, 3,6-diaminoacridine (50 µM), the force spectrum shows three main regions, with a large narrow distribution of force events occurring from nonspecific interactions. The unbinding events resulting from a single duplex and a double duplex are similar to the SSPE control, while the unbinding of double duplexes seems to be more dominant in this case. At 15 µM 3,6-diaminoacridine, the resulting unbinding forces appear similar to the measurements obtained in SSPE buffer, except for a new distribution centered at 0.50 nN, which has also been observed for dilute concentrations of the intercalator, ethidium bromide (Table 1). Although it is tempting to suggest that this new distribution may be a result of other binding geometries (16) Kubota, Y. K.; Motoda, Y.; Kuromi, Y.; Fujisaki, Y. Biophys. Chem. 1984, 19, 25.
5336
Langmuir, Vol. 18, No. 14, 2002
observed at low dye concentrations,17 we are mindful that the DNA sequence we chose may also result in partial overlaps that will make it harder to discriminate between the two. When we used the well-characterized DNA staining dye, ethidium bromide (EB), the results we obtained were very similar to those for 3,6-diaminoacridine. Unlike 3,6-diaminoacridine, which is sequence specific, ethidium bromide intercalates to DNA without sequence specificity.13 At a typical DNA staining concentration of EB (1.27 µM or 0.5 µg/mL),18 the DNA duplex unbinding forces clearly show a decrease compared to the DNA duplex measurements in the SSPE hybridizing buffer. Using a similar concentration of the ethidium bromide, Krautbauer et al.13 observed a shortened transition in single molecule DNA stretching experiments on a 1200 nm long DNA fragment (3500 bp). This was interpreted to reflect the prestretching and the unwinding of the double helix by intercalation. Higher forces observed at the end of the transition in their measurements were indicative of a stabilized structure presumably with the intercalated ethidium bromide. A 100-fold decrease in the concentration of EB showed results similar to those obtained at 1.27 µM EB. However, at a 105-fold dilution of EB (12.7 pM), unbinding forces resulting from a single duplex are observed, as well as an additional distribution centered (17) Schelhorn, T.; Kretz, S.; Zimmermann, H. W. Cell. Mol. Biol. 1992, 38, 345. (18) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning; Cold Spring Harbor Laboratory Press: New York, 1989; p 6.15.
Letters
at 0.48 nN, similar to what we observed with a very dilute concentration of 3,6-diaminoacridine. However, unlike the case with 3,6-diaminoacridine, we do not observe multiple DNA duplex interactions in the presence of EB. The experiments reported here demonstrate that force spectroscopy is a suitable technique to probe the effects of DNA binding intercalators. Using two intercalating compounds, 3,6-diaminoacridine and ethidium bromide, we have measured a significant reduction in the unbinding forces arising from a 16-mer DNA duplex. This reduction in force has been interpreted as a conformational change in the DNA duplex, such as the unwinding of the DNA helix, that the DNA has to undergo to accommodate the intercalating molecule. At very dilute concentrations of the intercalating agents, the unbinding forces measured are similar to measurements from the annealing buffer used as a control in our experiments. We believe that force spectroscopy is a promising technique that may be used to screen intercalating and groove binding drugs at the single-molecule level. Acknowledgment. This work was supported by grants from the National Science Foundation (CHE9871225), Research Corporation, and the Petroleum Research Fund, administered by the ACS (PRF 34464GB5). We are very grateful to Dr. Brady Cheek (MetriGenix, Inc., MD) for many helpful discussions. LA025669T
