pubs.acs.org/Langmuir © 2010 American Chemical Society
DNA-Molecular-Motor-Controlled Dendron Association Yawei Sun,† Huajie Liu,‡ Lijin Xu,§ Liying Wang,† Qing-Hua Fan,*,†and Dongsheng Liu*,‡ †
Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of Molecular Recognition and Function, Institute of Chemistry, Chinese Academy of Sciences (CAS), 100190 Beijing, China, ‡ Department of Chemistry, Tsinghua University, 100084 Beijing, China, and §Department of Chemistry, Renmin University of China, 100872 Beijing, China Received May 5, 2010. Revised Manuscript Received June 21, 2010 In this letter, we described a new strategy to study the macromolecule interactions rationally controlled by the movements of a DNA molecular motor. Two amphiphilic dendrons are covalently attached to the 30 and 50 ends of a pHdriven DNA motor, a 21-mer single-stranded DNA containing four stretches of cytosine-rich sequences. The resulting DNA-dendron conjugates were purified by polyacrylamide gel electrophoresis (PAGE), and their molecular weights were confirmed by MALDI-TOF. The reversible association-dissociation of the two DNA-attached dendrons controlled by the opening and closing of the DNA motor following pH changes was verified by circular dichroism spectroscopy and DNA stability studies in aqueous solutions. The results suggest that the DNA molecular motor may serve as a new platform for studying nonspecific and specific macromolecular interactions on the molecular level.
1. Introduction Over the past 10 years, several different types of DNA molecular motors have been reported, which have all shown the capability of producing well-defined, highly reversible nanoscale motions using different driving mechanisms.1-11 Among these, the DNA motor based on an i-motif structure is particularly attractive because it can undergo clean, swift, highly reversible extensioncontraction motions driven by environmental pH changes.11 In addition, the opening-closing of the DNA motor can produce forces (e.g., ∼10 pN) that are strong enough to move the endattached functional groups over a distance of ∼5 nm. This DNA motor has been exploited to construct a range of functional DNA (1) Alberti, P.; Bourdoncle, A.; Sacca, B.; Lacroix, L.; Mergny, J. L. Org. Biomol. Chem. 2006, 4, 3383–3391. (2) Bath, J.; Turberfield, A. J. Nat. Nanotechnol. 2007, 2, 275–284. (3) Liedl, T.; Sobey, T. L.; Simmel, F. C. Nano Today 2007, 2, 36–41. (4) Liu, H. J.; Liu, D. S. Chem. Commun. 2009, 2625–2636. (5) Yurke, B.; Turberfield, A. J.; Mills, A. P.; Simmel, F. C.; Neumann, J. L. Nature 2000, 406, 605–608. (6) Yan, H.; Zhang, X.; Shen, Z.; Seeman, N. C. Nature 2002, 415, 62–65. (7) Li, J. J.; Tan, W. Nano Lett. 2002, 2, 315–318. (8) Alberti, P.; Mergny, J. L. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 1569–1573. (9) Chen, Y.; Wang, M.; Mao, C. D. Angew. Chem., Int. Ed. 2004, 43, 3554– 3557. (10) Liedl, T.; Simmel, F. C. Nano Lett. 2005, 5, 1894–1898. (11) Liu, D. S.; Balasubramanian, S. Angew. Chem., Int. Ed. 2003, 42, 5734– 5736. (12) Wang, W. X.; Liu, H. J.; Liu, D. S.; Xu, Y.; Yang, Y.; Zhou, D. J. Langmuir 2007, 23, 11956–11959. (13) Liu, D. S.; Bruckbauer, A.; Abell, C.; Balasubramanian, C.; Kang, D. J.; Klenerman, D.; Zhou, D. J. J. Am. Chem. Soc. 2006, 128, 2067–2071. (14) Shu, W. M.; Liu, D. S.; Watari, M.; Riener, C. K.; Strunz, T.; Welland, M. E.; Balasubramanian, S.; McKendry, R. A. J. Am. Chem. Soc. 2005, 127, 17054–17060. (15) Wang, S.; Liu, H.; Liu, D.; Ma, X.; Fang, X.; Jiang, L. Angew. Chem., Int. Ed. 2007, 46, 3915–3917. (16) Mao, Y.; Liu, D.; Wang, S.; Luo, S.; Wang, W.; Yang, Y.; Ouyang, Q.; Jiang, L. Nucleic Acids Res. 2007, 35, e33. (17) Liu, H.; Zhou, Y.; Yang, Y.; Wang, W.; Qu, L.; Chen, C.; Liu, D.; Zhang, D.; Zhu, D. J. Phys. Chem. B 2008, 112, 6893–6896. (18) Xia, F.; Guo, W.; Mao, Y.; Hou, X.; Xue, J.; Xia, H.; Wang, L.; Song, Y.; Ji, H.; Ouyang, Q.; Wang, Y.; Jiang, L. J. Am. Chem. Soc. 2008, 130, 8345–8350. (19) Cheng, E. J.; Xing, Y. Z.; Chen, P.; Yang, Y.; Sun, Y. W.; Zhou, D. J.; Xu, L. J.; Fan, Q. H.; Liu, D. S. Angew. Chem., Int. Ed. 2009, 48, 7660–7663. (20) Stachowiak, J. C.; Yue, M.; Castelino, K.; Chakraborty, A.; Majumdar, A. Langmuir 2006, 22, 263–268.
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nanostructures, molecular devices, and advanced intelligent materials,12-22 including the controlled assembly of gold nanoparticles,12 and to drive the nanoscale bending of a microcantilever.13 According to an X-ray crystal structure, the 50 and 30 ends in the i-motif state are held only 0.8 nm apart.23 It is thus reasonable to anticipate that if two macromolecules are linked to both ends of the i-motif DNA strand then the interactions between the two macromolecules could be controlled on the molecular level by this DNA motor. To test this assumption and further extend our study on i-motif DNA-based molecular nanomachines, dendrimers, which have well-defined, highly branched nanoscale structures that may serve as protein mimics,24-26 have been chosen as the model macromolecules in this study. Several types of bioconjugates consisting of DNA and organic dendrons/dendrimers have been reported as promising building blocks for novel dendritic assemblies.27-33 However, in all of these constructs, the dendrons were attached to one end of the DNA strand only and the two dendrons/dendrimers were separated by a rigid, extended double-stranded DNA; therefore, direct interactions between the two dendritic units were not achieved.27 Herein, we synthesized a dendron i-motif DNA molecular motor system where two amphiphilic dendrons are (21) Lengerich, B.; Rawle, R. J.; Boxer, S. G. Langmuir 2010, 26, 8666–8672. (22) Zhao, W. T.; Lin, L.; Hsing, I. M. Langmuir 2010, 26, 7405–7409. (23) Kang, C.; Berger, I.; Lockshin, C.; Ratliff, R.; Moyzis, R.; Rich, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11636–11640. (24) Frechet, J. M. J.; Tomalia, D. A. Dendrimers and Other Dendritic Polymers; Wiley-VCH: New York, 2001. (25) Newkome, G. R.; Moorefield, C. N.; V€ogtle, F. Dendrimers and Dendrons: Concepts, Syntheses, Applications; Wiley-VCH: Weinheim, Germany, 2001. (26) Zeng, F.; Zimmerman, S. C. Chem. Rev. 1997, 97, 1681–1712. (27) Caminade, A. M.; Turrin, C. O.; Majoral, J. P. Chem.;Eur. J. 2008, 14, 7422–7432. (28) DeMattei, C. R.; Huang, B.; Tomalia, D. A. Nano Lett. 2004, 4, 771–777. (29) Choi, Y.; Mecke, A.; Orr, B. G.; Banaszak Holl, M. M.; Baker, J. R. Nano Lett. 2004, 4, 391–397. (30) Choi, Y. S.; Thomas, T.; Kotlyar, A.; Islam, M. T.; Baker, J. R. Chem. Biol. 2005, 12, 35–43. (31) Goh, S. L.; Francis, M. B.; Frechet, J. M. J. Chem. Commun. 2002, 2954– 2955. (32) Carneiro, K. M. M.; Aldaye, F. A.; Sleiman, H. F. J. Am. Chem. Soc. 2010, 132, 679–685. (33) Alemdaroglu, F. E.; Ding, K.; Berger, R.; Herrmann, A. Angew. Chem., Int. Ed. 2006, 45, 4203–4206.
Published on Web 07/07/2010
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Figure 1. Schematic presentation of the reversible switching of dendrimer conformation and dendron association driven by the i-motif DNA motor. (X is 50 -CCCTAACCCTAACCCTAACCC-30 , and Y is 30 -GATTGTGATTGTGATTG-50 .)
covalently attached to both ends of the DNA motor, which allows the study of the DNA-motor-controlled reversible dendron association/dissociation in aqueous solutions. Figure 1 schematically illustrates the use of the i-motif DNA molecular motor11 to control macromolecular interactions reversibly. An amphiphilic peripheral oligoethylene glycol (OEG) unitfunctionalized poly(arylether) dendron has been chosen as the model macromolecule in this study. This choice is based on the following factors: (a) Unlike the situation for water-soluble charged surface groups such as amines, there is minimal interaction between the electronegative DNA and the neutral OEGs on the dendron surface, ensuring that the measured macromolecule interactions are from the dendron units only and not from the DNA-dendron interactions. (b) The peripheral OEGs greatly improve the water solubility and biocompatibility of the target dendrimers. Under a slightly basic condition (pH 8.0), the two linked dendritic sectors are held about 5.8 nm apart because of the formation of a rigid extended double helix, namely, the open state.11 When the pH value decreases to 4.5, the DNA molecule change its conformation from an extended structure to the i-motif structure.11 This process brings the two amphiphilic dendrons into close proximity to each other, leading to dendron association, namely, the closed state. The hydrophobic interaction between the two dendrons in turn stabilizes the i-motif structure, resulting in a higher melting temperature (Tm).34-36 Therefore, a shift in the Tm value can be used to probe the strength of the interactions between the two linked dendrons. This study therefore presents a conceptually new approach to DNA nanotechnology in which the macromolecular interactions are rationally controlled by a DNA motor.
2. Experimental Section Materials. All chemicals were purchased from Sigma-Aldrich, and the organic solvents used were dried according to published methods. Water used in all experiments was Millipore Milli-Q deionized (15.6 MΩ). Oligonucleotides (DNA) were purchased from SBS Company (Beijing, China). Preparation of Dendritic DNA Motors. Dendritic active ester (D-NHS, 8 mmol) was dissolved in water and added to the amino-modified oligonucleotide (ODN) (20 nmol) in a total volume of 100 μL of 0.10 M sodium tetraborate buffer (pH 8.5). The reaction mixture was then shaken overnight at room tem(34) Blake, R. D.; Delcourt, S. G. Nucleic Acids Res. 1998, 26, 3323–3332. (35) SantaLucia, J.; Hicks, D. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 415– 440. (36) Shchepinov, M. S.; Mir, K. U.; Elder, J. K.; Frank-Kamenetskii, M. D.; Southern, E. M. Nucleic Acids Res. 1999, 27, 3035–3041.
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perature. Purification of the products was performed using a 10% denaturing polyacrylamide gel (400 V, 3.5 h) with trisborate/EDTA buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA) as the running buffer. Identification of the conjugates was achieved by UV shadowing. The respective bands were excised from the gel and incubated in Tris buffer (pH 7) overnight at 37 °C. CD Spectra of Dendritic DNA Motors. The CD spectra were collected on a Jasco-810 spectropolarimeter equipped with a temperature control unit at 20 °C. The spectrum of the duplex structure was collected using 1 μM oligonucleotides in a solution of 50 mM Tris and 50 mM NaCl buffer at pH 8.0 in a total volume of 400 μL in a quartz cuvette. CD Melting Spectra of Dendritic DNA Motors. The melting study of the closed state of the dendritic DNA motors was performed on a Jasco-810 CD spectropolarimeter equipped with a programmable temperature control unit at 285 nm. The melting points of the i-motif structure in MES buffer solution are around 63 °C from CD and UV measurements. From melting curves, 15 °C is chosen as the working temperature, which is high enough to stabilize the i-motif structure in our system. All spectra were collected by monitoring the sample from 15 to 95 °C at a temperature ramp rate of 0.5 °C/min.
Cycling of the Open and Closed States of the Dendritic DNA Motors. Multiple cycling of the dendritic DNA motors can be demonstrated by the alternating addition of HCl and NaOH.
3. Results and Discussion First, three amphiphilic dendrons bearing an activated ester group at the focal point were designed and synthesized by using a double-stage convergent strategy. (For details of the synthesis, see Supporting Information, SI.37) The obtained dendrons reacted with 30 and 50 amino-modified DNA to yield the corresponding dendritic DNA molecular motors (G1-5N3N-G1, G2-5N3N-G2, and G3-5N3N-G3, as shown in Figure 2 and Figure S1 in SI). For comparison purposes, the same dendrons were attached at only the 30 or 50 end of the same DNA sequence by using the same synthesis procedures, yielding the monodendron-modified DNA motors (3N-Gn and 5N-Gn, n = 1-3). All of these resulting dendritic DNA molecular motors were analyzed and purified by denaturing polyacrylamide gel electrophoresis (PAGE), and the molecular weight was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. All of the obtained molecular weights agreed well with the calculated values (Table S1 in SI). In addition, all of these dendritic DNA molecular motors are very soluble in water. With these dendritic DNA molecular motors in hand, we then studied the reversible conformational change of i-motif DNA in aqueous solution by using circular dichroism (CD) spectroscopy. The dendritic single-stranded DNA molecules were first premixed with an equal amount of strand Y in 50 mM PBS buffer (pH 8.0) to form the duplex at a dendritic DNA concentration of 1.5 μM. As shown in Figure 3, CD experiments clearly showed that secondgeneration dendritic DNA G2-5N3N-G2, for example, exhibited distinct characteristics of a B-form duplex DNA structure with a positive band near 275 nm, a negative band near 240 nm, and a crossover at 258 nm at pH 8.0, almost identical to those of the unmodified X/Y duplex.38 When the pH value was reduced to around 4.5 by the addition of HCl, the positive and negative bands shifted to 285 and 260 nm (Figure 3 and Figure S3 in SI), respectively, which are consistent with a typical i-motif structure.38 (37) Wooley, K. L.; Hawker, C. J.; Frechet, J. M. J. J. Am. Chem. Soc. 1991, 113, 4252–4261. (38) Berova, N.; Nakanishi, K.; Woody, R. W. Circular Dichroism: Principles and Applications; Wiley-VCH: New York, 2000.
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Figure 2. Molecular structures of amphiphilic dendrons (A) and dendritic i-motif DNA molecules (B).
Figure 3. CD spectra of dendritic DNA motor systems at pH 8.0 and 4.5.
These results clearly demonstrated that these DNA molecular motors still worked well after the attachment of the sterically demanding dendrons. To demonstrate the association of dendritic sectors driven by this DNA motor, we further investigated the thermal stabilities of these dendritic DNA molecules by measuring the melting temperature (Tm). As described above, at acidic pH (pH 4.5), the DNA molecule motor changed its configuration from the rigid, extended double-stranded helix to the four-stranded i-motif. As a result, this closing process could bring the two linked dendritic sectors in close proximity to each other, which might cause them to interact with each other. Considering that the dendron was surrounded by hydrophilic OEG chains, we proposed that, in the event of the linked two dendritic sectors meeting, the peripheral OEG chains could slide away to make room for the hydrophobic inner layers of the dendron to approach more closely, thus resulting in dendron association. Because of the hydrophobic effect, the i-motif DNA structure could be stabilized by this association process. As expected, there were striking increases of 14, 15, and 11 °C in the Tm of the first- to the third-generation dendritic DNA motors (Figure 4a 12498 DOI: 10.1021/la101802y
Figure 4. CD melting spectra of dendritic DNA motor systems at pH 4.5. (a) Dendrimers Gn-5N3N-Gn and (b) dendrimers Gn5N3N.
and Table S1) as compared to that of the unmodified DNA system (Tm = 63 °C, Table S1).34-36 Notably, the third-generation dendritic DNA molecule gave a slightly lower melting point than those of the DNA motors bearing smaller dendrons, which was probably due to the steric effect of the relatively bulky dendritic sectors. To confirm further that the high Tm value was correlated to the dendron association, the Tm of the monodendron-modified DNA motors, in which dendron association could Langmuir 2010, 26(15), 12496–12499
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4. Summary
Figure 5. Cycling of the open and closed states of the dendritic DNA G2-3N5N-G2 motor.
not occur, was also determined. As shown in Figure 4b and Figure S4 in SI, very similar melting points to that of the unmodified DNA system were observed for all six monodendron-modified DNA motors (Figure 4b and Figure S4 in SI). Therefore, these results indicated that the i-motif DNA molecular motor could control the interaction between the linked two molecules. Having demonstrated the dendron association, we finally investigated the reverse process, dendron dissociation driven by this DNA motor. When the pH value was adjusted to 8.0 again, the DNA molecule motor experienced a conformational change from the i-motif structure to the extended double-stranded helix as demonstrated by CD experiments. In addition, multiple cycling of the open and closed states of the dendritic DNA motors had also been demonstrated by the alternating addition of HCl and NaOH. As shown in Figure 5, the CD signals were monitored at 285 nm to reflect the conformational change of the motor system. Importantly, the switch was found to be repeated several times without obvious signal attenuation (Figure S5 in SI), and this opening and closing process occurred very quickly and could be completed in seconds.
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We have covalently attached organic dendrons onto a DNA molecular motor for the first time and have demonstrated a conceptually new approach to DNA nanotechnology in which a DNA motor actively controls the association/dissociation of the two attached dendritic macromolecules. The new finding achieved in this study suggests that this new concept may prove that it is feasible to use an i-motif motor as a general platform for studying nonspecific and specific macromolecular interactions on the molecular level. For example, we can attach proteins, polypeptides or other organic polymers onto the DNA motor and explore the reversible and controllable interactions in the future. In addition, this dendritic DNA molecular motor might have the potential for the development of a new kind of transport vehicle capable of responding to subtle changes in the physiological environment. Acknowledgment. We thank the National Basic Research Program of China (973 program, no. 2007CB935900), the Science 100 Program of CAS, and the National Natural Science Foundation of China (no. 20725309) for financial support. We also thank Professor Dejian Zhou and Professor Ming Li for beneficial discussions. Supporting Information Available: Molecular structures of dendritic DNA motors; synthesis and stained PAGE of dendritic DNA motors; MALDI-TOF MS and melting temperature of dendritic DNA motors; CD spectra of dendritic DNA motors; CD melting spectra of dendritic DNA motors; cycling of the open and closed states of dendritic DNA motors; synthesis and characterization of amphiphilic dendrons; 1H NMR, 13C NMR, and MS spectra of new compounds; and MALDI-TOF spectra of dendritic DNA motors. This material is available free of charge via the Internet at http://pubs.acs.org.
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