Surface-Relevant Regulable DNA Toroids Induced

Apr 3, 2009 - Dopamine (2-(3,4-dihydroxyphenyl)ethylamine), one of the important natural chemical neurotransmitters, has drawn plenti- ful attention t...
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Surface-Relevant Regulable DNA Toroids Induced by Dopamine Cunlan Guo, Zhelin Liu, Fugang Xu, Lanlan Sun, Yujing Sun, Tao Yang, and Zhuang Li* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, P. R. China ReceiVed: NoVember 18, 2008; ReVised Manuscript ReceiVed: February 24, 2009

Dopamine (2-(3,4-dihydroxyphenyl)ethylamine) is known as a natural chemical neurotransmitter and is also a cytotoxic and genotoxic molecule for cell apoptosis. In this work, the interaction of DNA with dopamine was investigated. Though the electrostatic interaction of DNA and dopamine was weak in aqueous solution, dopamine condensed circular pBR322 DNA into toroids on the mica surface cooperatively with ethanol. The formed DNA toroids came from the shrinking of DNA that was driven by ethanol-enhanced DNA-dopamine electrostatic interaction. The size of the DNA toroids could be modulated by varying the concentration of dopamine. This study offers useful information about the DNA condensation induced by monovalent cations and the sample preparation for AFM measurement and application. On the other hand, this work provides the potential strategies to prepare morphology and size controllable DNA condensates, which have valuable applications in gene transfection and nanotechnology. Introduction Dopamine (2-(3,4-dihydroxyphenyl)ethylamine), one of the important natural chemical neurotransmitters, has drawn plentiful attention to its role in physiology and the determination of content.1 Dopamine is also a cytotoxic and genotoxic molecule, which is relevant to neurodegenerative disorders such as Parkinson’s disease and ischemia-induced striatal damage. As dopamine has o-diphenol groups, its toxicity is attributed to the oxidation of dopamine to a reactive quinine moiety,2 and this oxidation of dopamine may induce DNA damage.3 Some researches show that dopamine could cause the apoptosis and death of cells, which is also related with its oxidation.4,5 Besides the oxidation, dopamine is positively charged in neutral condition, and thus it can interact with negatively charged DNA.6 This electrostatic interaction may modulate the morphology of DNA and result in DNA condensation. The morphology of DNA condensates is very sensitive to experimental conditions, such as reaction time, solution conditions, and condensing reagents. Sometimes, a slight change of experimental condition may cause apparently different results,7,8 especially when the interaction between DNA and condensing reagent is relatively weak. Since the positive charges on one dopamine molecule are less than the positive charges on one polyamine molecule such as spermine, the interaction of dopamine with DNA is weaker than that between polyamine and DNA. So the complex of dopamine and DNA can be easily affected by the outside conditions. DNA condensation can also be directed on a solid-liquid interface.9–12 The surface can be considered as a model similar to the interface inside or outside the cell. The process that DNA molecules adsorb onto the surface and subsequently condense with condensing reagents is relevant to the behavior of DNA in living cells, and is important in biophysics. For example, the DNA condensation caused by protamine on mica surface has been performed for the study of DNA condensation in the sperm * To whom correspondence should be addressed. Phone: +86 431 85262057. Fax: +86 431 85262057. E-mail: [email protected].

nucleus.9 The dynamics of DNA condensation at the solid-liquid interface has been investigated to understand the DNA compaction in cellular processes.10 So the investigation about the interaction of DNA and dopamine, especially their interaction on a surface, will be helpful for understanding the role of dopamine in cells. In present study, the interactions of DNA and dopamine were investigated using UV-visible absorption spectroscopy, gel electrophoresis, and circular dichroism (CD). Especially, the morphology characteristics of DNA with dopamine on mica surface were studied by atomic force microscopy (AFM). It was found that dopamine could induce the condensation of circular DNA and result in the formation of DNA toroids on the mica surface. The size of the resulted DNA toroids could be regulated mainly by tuning the concentration of dopamine. The formation mechanism of the toroids is proposed in this study. Experimental Section Chemicals and Materials. pBR322 DNA (4361 bp) and λ DNA (48 502 bp) were purchased from Fermentas International Inc. and were diluted to 50 ng/µL with ultrapure water before use. Dopamine hydrochloride was purchased from Alfa Aesar. 3-Aminopropyltriethoxysilane (APTES) was purchased from Aldrich. All of the other reagents were used as received without further purification, and all solutions were prepared using ultrapure water (>18.2 MΩ cm) sterilized at high temperature. Muscovite mica (KAll2(AlSi3)O10(OH)2, V-1 grade) was purchased from Linhe Street Commodity Marketplace (Changchun, China) and was cut into about 1 cm × 1 cm square pieces as AFM substrates. Both sides of the mica surface were freshly cleaved before use. Formation of DNA Toroids. The typical DNA toroids were prepared according to the following procedure. Aliquots of 20 µL solutions containing 5 ng/µL circular pBR322 DNA and 100 µM dopamine were thoroughly mixed and incubated at 25 °C for 30 min. Then, 20 µL of the mixture was deposited onto a freshly cleaved mica surface and settled for 8 min. The mica was gently rinsed with an anhydrous ethanol washing mode.

10.1021/jp810126f CCC: $40.75  2009 American Chemical Society Published on Web 04/03/2009

Interactions of DNA and Dopamine

Figure 1. Circular DNA reacted with 100 µM dopamine for 30 min and adsorbed on a freshly cleaved mica surface, then rinsed with anhydrous ethanol (a). As control experiments, circular DNA was adsorbed on bare mica surface, and then was washed with anhydrous ethanol (b). After washing, the samples were dried in air, and left sealed. (z scale is 10 nm.)

After that, it was blotted with filtered paper gently, then dried in air and left sealed for AFM imaging. To study the influence of experimental conditions, incubation time and dopamine concentration were investigated. Besides that, linear λ DNA was used instead of pBR322 DNA to study the effect of DNA nature; different washing modes (i.e., (a) anhydrous ethanol alone and (b) water alone, respectively) were also examined. Furthermore, silanized mica was used to study the influence of the surface properties. Silanization of Mica. The silanized solution was prepared by mixing APTES with ultrapure water. The final concentration of APTES was 0.05% by volume. Fifteen µL of this silanized solution was dropped onto freshly cleaved mica surface. After remaining for about 5 min, the mica was thoroughly rinsed with water, then blotted with filtered paper and dried in air. Instruments. All AFM experiments were carried out with a Digital Instruments Nanoscope IIIa (Santa Barbara, CA) in tapping mode. Silicon (Si) cantilevers with spring constants of 0.6-6.0 N/m below their resonance frequency (typically, 67-150 kHz) were used in the measurements. The AFM images were acquired in air under ambient conditions. Results were reported as mean ( standard deviation (number of measurements). The mobility shift assay of DNA-dopamine complexes was studied by agarose gel electrophoresis. Ten microliters of the complexes was loaded into 1.0 wt % agarose gel containing 1 × GeneFinder and ran at 100 V for approximately 40 min. Then the gel was photographed under UV light using a Vilber Lourmat Fluorescent Gel Imaging and Analysis System. UV-visible spectroscopy was performed with a Cary-500 UV-vis-NIR spectrometer (Varian, USA). CD measurement was performed on a JASCO J-810 circular dichroism spectropolarimeter. The concentrations of all compositions for UV-visible and CD measurements were 5 times that in AFM measurements for obtaining stronger signals. Results and Discussion Formation of DNA Toroids on Mica Surface. The formation of DNA toroids on mica surface was observed by AFM. As shown in Figure 1a, after 30 min’s incubation with dopamine and rinsing by anhydrous ethanol, circular DNA (pBR322 DNA) transformed into small toroids on the bare mica surface. However, when DNA alone was adsorbed on the bare mica surface without dopamine, no such toroids but only irregular clews were observed (Figure 1b). These results clearly indicated that dopamine could react with DNA and transform the morphology of circular DNA from loose circles to small toroids.

J. Phys. Chem. B, Vol. 113, No. 17, 2009 6069 Through measuring along the center of the circumferential lines, the average circumference of the DNA toroids was about 302 ( 22 nm (N ) 40) and the average height of the DNA toroids was about 2.07 ( 0.3 nm (N ) 40). The circumference was much smaller than that of the native DNA at B conformation, about 1530 nm.13,14 Meanwhile, the length of the resulted DNA toroids was also much shorter than that of the native DNA at A conformation.15 This further indicated that the change of DNA in the presence of dopamine was not attributed to the B-A conformation transition, but to the dopamine induced DNA condensation. Further careful examination of the AFM images revealed that the formed DNA toroids were not very regular. There were nodular and branchlike structures on these toroids (as indicated by a circle and an arrow respectively in Figure 1a). Besides that, there were many lower positions on the DNA toroids. These results indicated that the DNA condensation occurred in this experiment was incomplete. Varied Size and Morphology of the DNA Toroids in the Presence of Different Concentrations of Dopamine. Through changing the concentrations of dopamine, the size and morphology of DNA toroids varied in a certain range (Figure 2). Here for each concentration, we chose 12 h as reaction time. As shown in Figure 2a, when dopamine concentration was 500 µM, bigger nodular structures with shorter irregular thread formed instead of small toroids. The average height of the nodular structures was 5.50 ( 0.7 nm (N ) 40), and the average height of the irregular low thread was 0.78 ( 0.1 nm (N ) 40). By decreasing the concentration of dopamine in the mixed solutions, slightly irregular DNA toroids with different circumferences were obtained (Figure 2b-e), where their circumference increased and height decreased with the decreasing of dopamine concentration (Figure 2f). Note that the circumferences of the toroids with different concentrations of dopamine were all shorter than the circumference of native circular DNA. These dopamine-concentration-dependent experiments further confirmed the interaction between dopamine and DNA. The interaction enhanced with the increasing concentration of dopamine. These results from the dopamine-concentrationdependent experiments also provided a convenient approach to control the size and morphology of the formed DNA toroids. Moreover, when dopamine was 100 µM (Figure 2b), the smallest toroids were observed in our experiment. The average circumference was 285 ( 29 nm (N ) 40), and the average height was 2.08 ( 0.5 nm (N ) 40). The circumference here was a little shorter than the one reacted for 30 min (Figure 1a), which also indicated that the interaction of DNA and dopamine existed and could be promoted through the longer reaction time. Besides that, the formation mechanism of such DNA toroids could be deduced. There was mainly electrostatic interaction between negatively charged DNA and positively charged dopamine, which could neutralize part of the negative charges along the DNA backbone and consequently result in the DNA condensation in a certain extent. Meanwhile, the nodular structures existed along the DNA toroids showed that the groups on DNA strand interacted with the neighboring groups on the same strand, and thus led to the formation of irregular DNA toroids and the decreasing of DNA circumference (i.e., the DNA shrinking). This intrastrand interaction was different from the interstrand winding that was mentioned previously.16,17 The further discussion for this formation mechanism will proceed detailedly in the following sections.

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Figure 2. Typical tapping mode AFM images of the reaction products between circular DNA and different concentrations of dopamine: 500 µM (a); 100 µM (b); 50 µM (c); 7.5 µM (d); and 5 µM (e). DNA was incubated with dopamine for 12 h and then was adsorbed on a freshly cleaved mica surface. The samples were washed with anhydrous ethanol and air-dried. (z scale is 10 nm.) (f) Dependence of circumference and height of DNA toroids on the dopamine concentration.

Figure 3. (a) UV absorption spectra of circular DNA and dopamine complex in the presence of various concentrations of dopamine: 0 µM (black), 25 µM (red), 250 µM (green), and 500 µM (blue). The circular DNA concentration was 25 ng/µL. The orange curve was the UV absorption spectrum of free dopamine with the concentration of 500 µM. (b) The comparison of absorption between the circular DNA-dopamine complex and the sum values of circular DNA and dopamine.

Figure 4. (a) Mobility shift gel electrophoresis of circular DNA-dopamine complexes in the presence of different dopamine concentrations (concentrations of dopamine in lanes from left to right were 0, 5 µM, 7.5 µM, 50 µM, 100 µM, and 500 µM). The concentration of circular DNA was 5 ng/µL. (b) CD spectra of circular DNA-dopamine complexes and dopamine with different dopamine concentrations: 0 µM (black), 25 µM (red and magenta), 250 µM (green and olive), 500 µM (dark yellow and navy). The circular DNA concentration was 25 ng/µL.

Characterization of DNA-Dopamine Interaction in Solution. In order to reveal the cause for the formation of these DNA toroids, we studied the interaction of DNA and dopamine in solution. First, UV spectroscopy was used to evaluate the interaction between DNA and dopamine with different concentrations of dopamine (Figure 3). It was found that the maximum absorptions of free circular DNA and dopamine were centered

at 259 and 280 nm, respectively. With the increasing concentration of dopamine, the absorption of the circular DNA-dopamine complex increased and had a red shift (Figure 3a). Moreover, a detailed calculation of the absorptions among free circular DNA, free dopamine, and circular DNA-dopamine complex showed that the absorption sum of free circular DNA and free dopamine was a little less than the absorption of circular

Interactions of DNA and Dopamine

Figure 5. Typical tapping mode AFM image of linear DNA (λ DNA) reacted with 100 µM dopamine for 12 h and adsorbed on a freshly cleaved mica surface, then washed with anhydrous ethanol. After washing, the samples were dried in air. (z scale is 10 nm.)

DNA-dopamine complex (Figure 3b). This meant an indistinctive hyperchromic effect existed between circular DNA and dopamine, which suggested a weak interaction between them. Further investigation was performed through agarose gel electrophoresis (Figure 4a). DNA bands were observed for all samples and no distinguishable retardation was observed in the absence or presence of dopamine with different concentrations, which indicated that the interaction between DNA and dopamine did not make obvious DNA condensation. Furthermore, CD spectra in Figure 4b showed that the circular DNA molecules were in B-form with a positive peak centered at 271 nm and a negative peak centered at 245 nm (black line), which were consistent with previous report.18 With the addition of dopamine, both of the two peaks did not shift, which meant that the circular DNA did not have the conformation transition and remained its B-form in a large range of dopamine concentration. All of the obtained results showed that dopamine certainly interact with DNA in solution, but the interaction was considerably weak. And this weak interaction did not result in obvious conformation transition of DNA. Furthermore, combining with the AFM result that the circumference of the DNA toroids increased with the decreasing of dopamine concentration (Figure 2), it indicated that the DNA toroids observed on mica surface should not form in solution but form on the mica surface. Mechanism Studies on the Formation of DNA Toroids. The above studies have showed that the dopamine concentration and the reaction time could affect the size of the DNA toroids. In order to understand the formation mechanism of circular DNA and dopamine, some effect factors, such as DNA nature, washing mode, and the surface properties of mica, have been investigated. First, the effect of DNA nature has been considered. Here, the reaction of linear DNA (λ DNA) and dopamine was taken as a control experiment. When linear DNA reacted with 100 µM dopamine for 12 h (Figure 5) under the same experimental conditions as the circular DNA mentioned above (Figure 2b), side-by-side DNA structures, but no toroids were observed. Besides that, there were also some small nodular structures along the DNA strands (as indicated by an arrow in Figure 5), which also indicated the interaction between the neighboring groups on the same DNA strand. These nodular structures indicated that the formation mechanism of these morphologies by linear DNA and dopamine was the same as the formation mechanism of DNA toroids. Therefore, only circular DNA molecules shrinking on mica surface yielded the DNA toroids, while linear DNA would not form the toroids. Besides these results mentioned above, the rinsing solvent, ethanol, also played an important role in the formation of DNA

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Figure 6. Circular DNA reacted with 100 µM dopamine for 30 min and adsorbed on a freshly cleaved mica surface, then washed with water (a). As a control experiment, free circular DNA was adsorbed on bare mica surface and then was washed with water (b). After washing, the samples were dried in air, and left sealed. (z scale is 10 nm.)

Figure 7. Circular DNA reacted with 100 µM dopamine for 30 min and adsorbed on the APTES modified mica surface, then washed with water (a) and anhydrous ethanol (b). After washing, the samples were dried in air and left sealed. (z scale is 10 nm.)

toroids in the presence of dopamine. In order to further prove it, water was chosen as controlling. When the circular DNA-dopamine complex was rinsed with water (Figure 6a), no DNA toroids but rigid threads with branched structures were observed. The average height of the rigid threads was about 0.97 ( 0.07 nm (N ) 40), which was lower than the sample that was washed with ethanol. The presence of ethanol could lower the dielectric constant of solution and change the electrostatic environment of DNA molecules, which could increase the electrostatic interactions between DNA and the bound molecules19 and thus promote the DNA condensation. In our present study, through rinsing with ethanol, dopamine and ethanol cooperatively reacted with circular DNA on the mica surface in a very short time, which induced the formation of DNA toroids. On the other hand, when free circular DNA was rinsed with water, mainly a few dots were found on the mica surface (Figure 6b). Compared with the result of Figure 6a, this suggested that there were some dopamine molecules that existed between DNA and mica surface. Dopamine could adsorb on the mica surface, which was possible because recent reports revealed that dopamine could strongly react with and adsorb on many substrates in the covalent and noncovalent form.20,21 These dopamine molecules could immobilize DNA molecules and prevent the motion of DNA on the mica surface. The immobilization and prevention might also work in ethanolrinsing case, though the shrinking that cooperatively produced by dopamine and ethanol was dominating. Additionally, the APTES-modified mica was used to investigate the competition between immobilization and shrinking, and to further understand the mechanism. As we know, electrostatic interactions exist between the positively charged NH2 groups on the APTES-modified mica surface and negatively

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SCHEME 1: Proposed Mechanism for the Formation of DNA Toroids on the Mica Surface Induced by Dopamine

charged phosphate of DNA.22 Thus, the APTES-modified mica surface could fix DNA molecules relatively firm and make DNA keep its initial morphologies in solution from changing by rinsing solvent. Figure 7 showed the morphologies of DNAdopamine complexes which were adsorbed on APTES-modified mica surface and rinsed with different solvents. When the adsorbed complex was rinsed with water and dried in air, natural extended circular DNA molecules were observed, but no toroids were found (Figure 7a). The average height of the DNA strands was 0.47 ( 0.03 nm (N ) 40), which was in agreement with the native height of the double-stranded DNA measured by tapping mode AFM in air.23,24 Comparing Figure 6a with 7a showed that the immobilization of circular DNA only by dopamine was quite weak. After the adsorbed complex was rinsed with ethanol (Figure 7b), the loosely clew-like circular DNA was mainly observed on the APTES-modified mica surface instead of DNA toroids. The average height of the loosely clew-like molecules was 0.67 ( 0.04 nm (N ) 40), which was close to the native height of the double-stranded DNA. Besides that, some tori (one was indicated with an arrow) were also observed among the loose-crowded molecules, which seemed to contain dots on them. The average circumference of the tori was 305 ( 36 nm (N ) 35), and the average height of the tori was 1.37 ( 0.19 nm (N ) 40). These results indicated that the immobilization of DNA by APTES strongly prevented the shrinking of extended DNA that was induced by ethanol enhanced DNA-dopamine electrostatic interaction. Dopamine molecules have been proved previously to immobilize DNA. The immobilization by dopamine similarly prevented the shrinking of DNA molecules, but this prevention was relatively weak. Therefore, at the low concentrations of dopamine, DNA did not form irregular clews but larger toroids due to the dual contradictory roles of dopamine for DNA condensation and immobilization (Figure 2). However, when dopamine was as high as 500 µM, the condensing interaction was much greater than the prevention, so bigger nodular structures instead of toroids were obtained (Figure 2a). The results obtained from the APTES-modified mica surface further indicated that the

DNA toroids formed through the movement on the bare mica surface that was driven by dopamine and ethanol cooperatively. According to all the obtained results above, a reasonable mechanism has been proposed to explain the formation of DNA toroids on the mica surface induced by dopamine (Scheme 1). Dopamine could interact with DNA in aqueous solution, but the interaction was too weak to change the DNA conformation significantly (I). When the DNA-dopamine complex was dropped onto the bare mica surface (step a), a part of dopamine that interacted with mica adsorbed and fixed DNA on the mica surface gently, and a part of dopamine interacted with DNA mainly in the mode of electrostatic interaction (II). When ethanol was used to rinse the mica surface (step b), it lowered the dielectric constant and then enhanced the electrostatic interactions between DNA and dopamine. The increase in electrostatic interactions that were cooperatively aroused by dopamine and ethanol led circular DNA to shrink. On the other hand, dopamine adsorbed onto the mica could immobilize DNA as molecular glue and subsequently inhibit the ethanol-induced DNA movement slightly (III). This competition resulted in the final formation of DNA toroids but not the DNA dots or clews (IV). The size of the formed DNA toroids could be adjusted through changing the dopamine concentration. Conclusion In summary, we studied the interaction between DNA and dopamine. The results indicated that DNA reacted with dopamine mainly through electrostatic interaction. The interaction of DNA and dopamine was weak, and DNA held its natural extended conformation in aqueous solution with dopamine. More importantly, circular DNA condensed into toroids on the bare mica surface. The size and morphology of the formed toroids could be adjusted by varying the concentration of dopamine. Through the comparisons among different effect factors, it showed that the DNA toroids were formed by the shrinking of extended DNA on the mica surface, which was mainly induced by ethanol-enhanced DNA-dopamine electrostatic interaction. This study displayed the complicated behavior

Interactions of DNA and Dopamine of DNA at the solid-liquid interface. It also provided methods to control the size and morphology of the DNA condensates, which might be valuable for gene delivery in medicine science and nanotechnology in material science. In addition, it supplied useful notes on the sample preparation for those using AFM as tools. Acknowledgment. This work was supported by the National Natural Science Foundation of China (20775077) and the Chinese Academy of Sciences (KJcx2-YW-H11). We are grateful to Gaiping Li and Yan Du for the help of our experiments. We especially appreciate our three anonymous referees for their valuable comments and suggestions. References and Notes (1) Tsunoda, M. Anal. Bioanal. Chem. 2006, 386, 506–514. (2) Stokes, A. H.; Hastings, T. G.; Vrana, K. E. J. Neurosci. Res. 1999, 55, 659–665. (3) Yamada, K.; Shirahara, S.; Murakami, H.; Nishiyama, K.; Shinohara, K.; Omura, H. Agric. Biol. Chem. 1985, 49, 1423–1428. (4) Simantov, R.; Blinder, E.; Ratovitski, T.; Tauber, M.; Gabbay, M.; Porat, S. Neuroscience 1996, 74, 39–50. (5) Kang, C. D.; Jang, J. H.; Kim, K. W.; Lee, H. J.; Jeong, C. S.; Kim, C. M.; Kim, S. H.; Chung, B. S. Neurosci. Lett. 1998, 256, 37–40. (6) Liu, J.; Wang, Z. H.; Luo, G. A.; Li, Q. W.; Sun, H. W. Anal. Sci. 2002, 18, 751–755. (7) Vilfan, I. D.; Conwell, C. C.; Sarkar, T.; Hud, N. V. Biochemistry 2006, 45, 8174–8183. (8) Conwell, C. C.; Vilfan, I. D.; Hud, N. V. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 9296–9301.

J. Phys. Chem. B, Vol. 113, No. 17, 2009 6073 (9) Allen, M. J.; Bradbury, E. M.; Balhorn, R. Nucleic Acids Res. 1997, 25, 2221–2226. (10) Ono, M. Y.; Spain, E. M. J. Am. Chem. Soc. 1999, 121, 7330– 7334. (11) Fang, Y.; Hoh, J. H. Nucleic Acids Res. 1998, 26, 588–593. (12) Zhang, C.; van der Maarel, J. R. C. J. Phys. Chem. B 2008, 112, 3552–3557. (13) Liu, Z. G.; Li, Z.; Zhou, H. L.; Wei, G.; Song, Y. H.; Wang, L. Microsc. Res. Tech. 2005, 66, 179–185. (14) Ellis, J. S.; Abdelhady, H. G.; Allen, S.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. J. Microsc.-Oxford 2004, 215, 297–301. (15) Zheng, J. P.; Li, Z.; Wu, A. G.; Zhou, H. L. Biophys. Chem. 2003, 104, 37–43. (16) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334–341. (17) Hud, N. V.; Vilfan, I. D. Annu. ReV. Biophys. Biomol. Struct. 2005, 34, 295–318. (18) Grguric-Sipka, S. R.; Vilaplana, R. A.; Pe´rez, J. M.; Fuertes, M. A.; Alonso, C.; Alvarez, Y.; Sabo, T. J.; Gonza´lez-Vı´lchez, F. J. Inorg. Biochem. 2003, 97, 215–220. (19) Mel’nikov, S. M.; Khan, M. O.; Lindman, B.; Jonsson, B. J. Am. Chem. Soc. 1999, 121, 1130–1136. (20) Lee, H.; Scherer, N. F.; Messersmith, P. B. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 12999–13003. (21) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science 2007, 318, 426–430. (22) Hu, J.; Wang, M.; Weier, H. U. G.; Frantz, P.; Kolbe, W.; Ogletree, D. F.; Salmeron, M. Langmuir 1996, 12, 1697–1700. (23) Lyubchenko, Y. L.; Shlyakhtenko, L. S. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 496–501. (24) Thundat, T.; Allison, D. P.; Warmack, R. J. Nucleic Acids Res. 1994, 22, 4224–4228.

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