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Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B–A transition in solution

Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B–A transition... Published online August 2, 2006 3670–3676 Nucleic Acids Research, 2006, Vol. 34, No. 13 doi:10.1093/nar/gkl513 Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B–A transition in solution Xi Li, Yinghua Peng and Xiaogang Qu* Division of Biological Inorganic Chemistry, Key Laboratory of Rare Earth Chemistry and Physics, Graduate School of the Chinese Academy of Sciences, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China Received May 5, 2006; Revised June 6, 2006; Accepted July 5, 2006 single stranded DNA self-assembles into a helical structure ABSTRACT around individual carbon nanotubes. Since carbon nanotube– Single-walled carbon nanotubes (SWNTs) have DNA hybrids have different electrostatic properties that been considered as the leading candidate for nano- depend on the diameter of the nanotubes and electronic prop- device applications ranging from gene therapy erties, they can be separated and sorted using anion exchange and novel drug delivery to membrane separations. chromatography (11,12). Carbon nanotubes are able to con- The miniaturization of DNA-nanotube devices for dense double stranded plasmid DNA to varying degree and exhibit upregulation of marker gene expression over naked biological applications requires fully understanding DNA using a mammalian (human) cell line, a nanotube- DNA-nanotube interaction mechanism. We report based gene-delivery vector has been reported (16). In a recent here, for the first time, that DNA destabilization report, a piece of double-stranded DNA wrapped on the sur- and conformational transition induced by SWNTs face of a single-walled carbon nanotube can serve as sensors are sequence-dependent. Contrasting changes for in living cells (17) and the heart of the new optical detection SWNTs binding to poly[dGdC]:poly[dGdC] and system is based on the transition of DNA secondary structure poly[dAdT]:poly[dAdT] were observed. For GC from the native, right-handed ‘B’ form to the alternate, left- homopolymer, DNA melting temperature was handed ‘Z’ form which was modulated by metal ions. decreased 40 C by SWNTs but no change for Therefore, it is important and fundamental to understand AT-DNA. SWNTs can induce B–A transition for GC- the interaction mechanism of SWNT with double-stranded DNA but AT-DNA resisted the transition. Our circular DNA for nanodevice application. dichroism, competitive binding assay and triplex In this report, SWNTs DNA binding mode, binding preference and the impact on DNA stability and conformation destabilization studies provide direct evidence that were studied. Contrasting changes for SWNTs binding to SWNTs induce DNA B–A transition in solution and poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were they bind to the DNA major groove with GC observed. We report here, for the first time, that DNA preference. condensation, destabilization and conformational transition induced by SWNTs are sequence-dependent. Our circular dichroism (CD), competitive binding assay and triplex INTRODUCTION destabilization studies provide direct evidence that SWNTs Single-walled carbon nanotubes (SWNTs) have been con- induce DNA B–A transition in solution and they bind to sidered as the leading candidate for nanodevice applications the DNA major groove with GC preference. The sequence- because of their one-dimensional electronic band structure, dependent condensation and B–A transition by SWNTs molecular size, biocompatibility, controllable property of shed light on the design of miniature of optical devices and conducting electrical current and reversible response to bio- label-free detection of specific genes. chemical reagents (1–6). These potential applications range from gene therapy and drug delivery to membrane separa- tions (4–16). Among the molecules that can non-covalently MATERIALS AND METHODS bind to the surface of SWNTs, DNA has been the research SWNTs (f ¼ 1.1nm, purity >90%) were purchased from focus (7–16), which adsorbs as a single-strand or double- Aldrich and purified as described previously by sonicating strand complexes. By screening a library of oligonucleotides, SWNTs in a 3:1 v/v solution of concentrated sulfuric acid previous reports have shown that a particular sequence of *To whom correspondence should be addressed. Tel: 86 431 526 2656; Fax: 86 431 5262656; Email: [email protected] 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2006, Vol. 34, No. 13 3671 (98%) and concentrated nitric acid (70%) for 24h at 35–40 C striking differences were observed among these DNA and washed with water, leaving an open hole in the tube side molecules (Figure 1D–F). SWNTs condense DNA depending and functionalized the open end of SWNTs with carboxyl on DNA GC content: GC homopolymer, GC-DNA was con- group to increase their solubility in aqueous solution (2). densed (23) as a network (Figure 1D). For calf thymus The stock solution of SWNTs (0.15 mg mL ) was obtained DNA (ct-DNA) and AT-DNA, they looked like forming by sonicating the SWNTs for 8 h in pH 7.0 aqueous solution. DNA-wrapped complexes and condensed slightly Calf thymus DNA (ct-DNA) was obtained from Sigma and (Figure 1E and F) showing that GC-DNA was more easily purified as described earlier (18). Poly[dGdC]:poly[dGdC] condensed than AT-DNA and ct-DNA. Premilat et al. have (GC-DNA), poly[dAdT]:poly[dAdT] (AT-DNA), polydA, measured the major groove width of GC-DNA (1.35 nm) and polydT were purchased from Pharmacia. The concentra- and AT-DNA (1.75 nm) through fiber X-ray diffraction tion of ct-DNA, GC-DNA and AT-DNA were determined by (26,27). SWNTs (f ¼ 1.1nm) we used were modified with ultraviolet absorbance measurements using the extinction carboxyl group. Carboxyl groups at the open end of 1 1 1 coefficient: e ¼ 12 824 M cm , e ¼ 16 800 M SWNTs greatly increased their water solubility and may 260 262 1 1 1 cm , e ¼ 13200 M cm , respectively (18). Triplex impact DNA binding to the modified nanotube surface. DNA (polydA:[polydT] ) was prepared as described previ- Based on SWNTs size, hydrophobic property and their ously (19–21). Ethidium bromide (EB), hoechst 33258 and improved solubility, SWNTs should not bind to DNA daunomycin (DM) were purchased from Sigma, methylene minor grooves due to the narrower groove width. Alternat- green was purchased from Aldrich and were used without fur- ively, SWNTs may bind to the major groove and would fit ther purification. Their concentrations were determined by better to GC-DNA major groove because AT-DNA major absorbance measurements using the extinction coefficient: groove is too wide for SWNTs binding. Hansma and Kankia 1 1 1 1 e ¼ 5600 M cm , e ¼ 42 000 M cm , have reported that condensation of DNA by metal ions is 480 338 1 1 e ¼ 11 500 M cm for EB, Hoechst 33 258 and DM, sequence dependent owing to the difference of GC and AT respectively (22). All the experiments were carried out in sequence in their extent of dehydration (23,28,29). GC-rich regions are being more heavily dehydrated than AT-rich Tris buffer (10 mM Tris, pH ¼ 7.1) unless stated otherwise. In sodium iodide fluorescence quenching experiments, the regions, this can be the reason why GC-DNA was easily con- ionic strength was kept constant. densed by SWNTs and this will be further discussed in our An AFM (Nanoscope IIIa, Digital Instruments, Santa Bar- CD studies. SWNTs, as gene-delivery vector (16), can take bara, CA) was used to image all DNAs in the presence or advantage to select highly GC-content gene for delivery absence of SWNTs. The sample solution was deposited and this needs to be verified in various gene expression onto a piece of freshly cleaved mica and rinsed with water systems. and dried before measurements (23). Tapping mode was used to acquire the images under ambient condition. CD SWNTs selective destabilization of DNA spectra were measured at 20 C on a JASCO J-810 spectro- polarimeter with a computer-controlled water bath (24). Figure 2 shows DNA UV melting profiles in the absence or The optical chamber of CD spectrometer was deoxygenated presence of SWNTs. Contrasting changes for SWNTs binding with dry purified nitrogen (99.99%) for 45 min before use to GC-DNA and AT-DNA were observed. It is obvious that and kept the nitrogen atmosphere during experiments. GC-DNA and ct-DNA became unstable in the presence of Three scans were accumulated and automatically averaged. SWNTs. Melting temperature T decreased 40 C for GC- Absorbance measurements and melting experiments were DNA when SWNTs at 25 mg/ml (Figure 2A). The absorption made on a Cary 300 UV/Vis spectrophotometer, equipped after 80 C decreased showing the strong interaction of single- with a Peltier temperature control accessory (25). All UV/ strand DNA with SWNTs (11,12). Since the melting temper- Vis spectra were measured in 1.0 cm path length quartz ature of GC-DNA in the presence of 15 mgml or higher SWNTs concnentration was much lower than 60 C, it cuvettes with the same concentration of SWNTs aqueous seems unlikely that the destabilization was due to pH change solution as the reference. Absorbance changes at 260 nm with temperature. On the contrary to GC-DNA and ct-DNA, versus temperature were collected at a heating rate of SWNTs did not influence AT-DNA stability even at high 0.5 C min for DNA melting experiments. Primary data concentration of SWNTs (Figure 2C). At 20 mM NaCl, were transferred to the graphics program Origin for plotting similar trend for SWNTs bound to DNA was observed and analysis. Fluorescence experiments were carried out on a JASCO FP-6500 spectrofluorometer at 20 C (24). (data not shown). We also studied SWNTs bound to triplex DNA at high salt conditions which will be addressed in triplex destabilization section. These results showed that the binding preference of SWNTs was: GC-DNA>ct-DNA>AT- RESULTS AND DISCUSSION DNA, consistent with the AFM and CD results which will SWNT inducing sequence-dependent be addressed next. DNA condensation AFM studies showed that DNA condensed when they bound SWNTs making DNA B–A transition in solution to carboxyl-modified SWNTs (2) and the condensation was dependent on DNA composition. In the absence of SWNTs, As DNA bound to carboxyl-modified SWNTs, various inter- linear GC-DNA, AT-DNA and natural DNA, purified calf actions of DNA bases and backbone with SWNTs, such as hydrophobic interactions, van der Waals, electrostatic inter- thymus DNA (ct-DNA) were observed (Figure 1A–C). actions, can take place (15). The strong interactions between When they bound with carboxyl-modified SWNTs, however, 3672 Nucleic Acids Research, 2006, Vol. 34, No. 13 Figure 1. DNA AFM images in the absence or presence of SWNTs: (A) GC-DNA alone; (B) ct-DNA alone; (C) AT-DNA alone; (D) GC-DNA + 40 mgml 1 1 1 SWNTs; (E) ct-DNA + 40 mgml SWNTs; (F) AT-DNA + 40 mgml SWNTs. The DNA concentration used in all experiments was 19.5 mgml . All the AFM images are captured on freshly cleaved mica. The image for (A–C) is 500 nm · 500 nm and the same scale bar. The image for (D–F) is 1 mm · 1 mm and with the same scale bar. SWNTs and DNA can disturb DNA hydration layer, even make DNA B–A transition in solution, consistent with DNA structure (22,29). previous molecular dynamics simulation results which show CD spectra showed these DNA molecules were in B-form B–A transition when DNA encapsulated in carbon nanotube (Figure 3) with a positive band near 270 nm and a negative or on gold surface (7,9). The induced B to A transition was band near 250 nm in the absence of SWNTs (18,24). Upon due to SWNTs bound to the major groove resulting in deep- addition of SWNTs (Figure 3A), the canonical B form of ening and narrowing the major groove while widening the GC-DNA altered with a positive band near 258 nm and a minor groove, which was coincident with the previous simu- negative band near 242 nm indicating that B–A transition lation results (33). (28–33) occurred. The transition was cooperative and the Calf thymus DNA (ct-DNA, 42% GC and 58%AT) was transition midpoint was at 10 mg/ml SWNTs (Figure 4). CD induced to A-form by SWNTs but not as easily as GC-DNA spectroscopy provided the direct evidence that SWNTs could while AT-DNA persisted in B-form (Figure 3B and C), Nucleic Acids Research, 2006, Vol. 34, No. 13 3673 1.0 A-Form 0.5 −1 +25 µg ml DNA alone SWNTs 0.0 30 45 60 75 90 −2 1.0 B-Form −4 CD258nm 0.5 CD252nm −6 0.0 −8 30 40 50 60 70 80 0 5 10 15 20 25 1.0 Conc (SWNT), µg/ml Figure 4. Plot of CD intensity versus concentration of SWNTs. CD intensity 0.5 at 258 nm (solid circles) and at 252 nm (open circles). The data were adopted from Figure 3A. 0.0 showing that B–A transition was dependent on the G-C con- 30 40 50 60 70 80 tent of the DNA helix (28–33). Ivanov and Krylov (34) have Temperature (°C) reviewed the cooperative character of B–A transition and determined the cooperative width of B–A transition with Figure 2. UV melting profiles of DNA: (A) poly[dGdC]:poly[dGdC], three different methods, and confirm that the cooperative (B) ct-DNA, (C) poly[dAdT]:poly[dAdT] in the absence or presence of width of B–A transition for DNA with mixed sequence is SWNTs. From right to left: 0, 1, 5, 10, 15, 20, 25 mgml SWNTs in pH ¼ 7 in the range of 10–30 bp. Since GC-DNA was more easily solution. Normalized absorption changes at 260 nm were plotted against temperature. The data were collected at a heating rate of 0.5 C min . Details condensed by SWNTs, the width of the transition for GC see Materials and Methods. homopolymer should be lower than that for the DNA with mixed sequence (31), such as ct-DNA. Since the water activ- ity is an apparent driving force for B–A transition and the 5 A-Form water activity (31) of GC-rich region (81.2) is lower than AT-rich region (81.5), GC-DNA undergoes the B to A trans- −5 ition most easily, whereas AT-DNA resists the B to A trans- B-Form ition. Previous studies show that GC homopolymer have a −10 stronger tendency for aggregation than AT homopolymer, −15 and GC homopolymer can undergo B–Z–A transition by reducing water activity (35). Under the usual experimental 10 conditions for B–A transition, GC homopolymer will aggreg- ate (36) while AT homopolymer would remain in the B-form even at conditions which normally favors the A-form (36,37). These results further support that GC homopolymer was more easily condensed by SWNTs than AT homopolymer as shown −5 in our AFM studies. A-DNA is biologically relevant and has 11 bp per helical turn, base pairs are tilted to 20 C with respect to the helical axis (38), the grooves are not as deep as those in B-DNA, the 0 0 sugar pucker is C3 endo compared with C2 endo for B-DNA. Like B–Z DNA transition (17,18,24,39,40), the transition −3 from the B-DNA double helix to the A-form is essential for biological function (28–33), as shown by the existence of 200 220 240 260 280 300 320 the A-form in many protein–DNA complexes, increasing the Wavelength (nm) fidelity of DNA and RNA synthesis and protection from DNA damage. Figure 3. CD spectra of DNA. (A) poly[dGdC]:poly[dGdC] (B) ct-DNA (C) poly[dAdT]:poly[dAdT] in the absence (black) or presence of SWNTs: Fluorescence competitive binding assay and triplex SWNTs 1 (red), 5 (green), 10 (blue), 15 (cyan), 20 (magenta) and 25 mgml (yellow) in Tris buffer (pH ¼ 7.1) at 20 C. CD spectra were measured on a DNA destabilization by SWNTs JASCO J-810 spectropolarimeter with a computer-controlled water bath as It is well known that EB and DM can intercalate into DNA described in Materials and Methods. Three scans were accumulated and automatically averaged. through the minor groove and Hoechst 33 258 is a classical Normalized A Ellipticity (mDegree) CD Intensity 3674 Nucleic Acids Research, 2006, Vol. 34, No. 13 A 30 A 500 550 600 650 700 400 450 500 550 600 650 C 0 50 100 150 200 Concentration of Iodide (mM) Figure 6. Plot of fluorescence intensity of fluorephore-DNA (squares) or fluorephore-DNA–SWNTs (circles) versus NaI concentration. The ionic strength was kept constant: (A)10 mM EB (Excitation wavelength: 480 nm, 500 550 600 650 700 Emission wavelength: 590 nm Slit: 5 nm); (B)10 mM Hoechst 33 258 (Excitation wavelength: 355 nm, Emission wavelength: 450 nm Slit: 3 nm); Wavelength (nm) (C)10 mM DM (Excitation wavelength: 480 nm, Emission wavelength: 565 nm Slit: 10 nm). Experimental details described in the experimental section. 300 D 590nm 450nm 10 565nm −3 −2 −10 −10 Log (Conc(SWNT), µg/ml) 300 400 500 600 700 Wavelength (nm) Figure 5. Fluorescence emission spectra of (A)1 mM EB (Excitation wavelength: 480 nm, Slit: 10 nm); (B)1 mM Hoechst 33 258 (Excitation wavelength: 355 nm, Slit: 3 nm); (C)1 mM DM (Excitation wavelength: 480 Figure 7. Loss of CD signal from calf thymus DNA-methylene green nm, Slit: 10 nm): 1mM fluorophore (EB, Hoechst 33 258, DM) alone (black complex upon SWNT–DNA association. Methylene green (black); calf curves), 1 mM fluorophore + 30 mgml CT-DNA (open circles), 1 mM thymus DNA (red); calf thymus DNA-methylene green (green); calf thymus 1 1 fluorophore + 30 mgml CT-DNA + 1 mgml SWNTs (upper triangles). DNA-methylene green after association with SWNTs at binding ratio r ¼ 0.2 (D) Fluorescence intensity as a function of concentration of SWNTs: 1 mM (blue). Calf thymus DNA was 50 mM in bp. CD spectra were measured at 1 1 EB + 30 mgml CT-DNA (squares), 1 mM Hoechst 33 258 + 30 mgml 20 C on a JASCO J-810 spectropolarimeter with a computer-controlled water CT-DNA (circles), 1 mMDM + 30 mgml CT-DNA (triangles). The bath as described in the experimental section. Three scans were accumulated experiments were carried out in Tris buffer (pH ¼ 7.1) at 20 C. and automatically averaged. DNA minor groove binder. When bound to DNA, the fluores- not competitively bind to the same sites. To further identify cence of EB or Hoechst is greatly enhanced, and DM fluores- their different binding sites, we carried out NaI quenching cence is strongly quenched. With this in mind, if SWNTs experiments. Iodide ions cannot quench the fluorescence of competitively bind to the same sites of DNA as EB, Hoechst these dye molecules when they are bound to DNA (43). If and DM, the fluorescence of EB and Hoechst would decrease SWNTs could replace these molecules, their fluorescence and the fluorescence of DM would increase because the should be quenched. Figure 6 showed that the fluorescence strong binding of SWNTs with DNA should exclude was not quenched by iodide at all. However, we found that these DNA binders out of their binding sites. The fluores- SWNTs could exclude methylene green, a proven DNA cence competitive binding assay has been widely used major groove binder (44,45), out of DNA. Figure 7 showed to establish DNA binding mode (41–43). As shown CD spectral changes in the presence of SWNTs. For methyl- in Figure 5A–C, their fluorescence hardly changed, even ene green, there was no CD signal in our experimental condi- titrated by SWNTs (Figure 5D), showing that SWNTs do tions. When methylene green bound to DNA, three induced Fluorescence Intensity (a.u) Fluorescence Intensity (a.u) I I I 565nm 450nm 590nm Ellipticity (mDegree) Nucleic Acids Research, 2006, Vol. 34, No. 13 3675 1.0 therapeutic agents, and because of the possible relevance of H-DNA structures in biology system (19–21). However, 0.8 SWNTs selective destabilization of triplex DNA has not been reported. Based on previous computer simulation results 0.6 of SWNTs binding to duplex DNA in the major groove (14) 0.4 and our melting, CD, and competitive binding data, SWNTs probably bound to duplex DNA major groove and had GC 0.2 preference. 0.0 30 40 50 60 70 80 90 CONCLUSIONS Temperature (°C) SWNTs can cause sequence-dependent DNA condensation Figure 8. UV melting profiles of triplex DNA polydA (polydT) in the and strongly destabilize GC-DNA. Contrasting changes for 1 1 absence (black) or presence of SWNTs: SWNTs 1 mgml (red); 2 mgml 1 1 SWNTs binding to GC-DNA and AT-DNA were observed. (green); 5 mgml (blue); 10 mgml (cyan) in Tris buffer (10 mM Tris, Our CD, competitive binding and triplex destabilization stud- 200 mM NaCl, pH ¼ 7.1). Normalized absorption changes at 260 nm were plotted against temperature. The data were collected at a heating rate of ies provide direct evidence that SWNTs induce DNA B–A 0.5 C min . transition in solution and they bind to the DNA major groove with GC preference. CD signals characteristic of bound methylene green around 310 nm, 430 nm, 650 nm were observed (44,45). With addi- ACKNOWLEDGEMENTS tion of SWNTs, the induced CD intensity was decreased and even disappeared, typical data was shown in Figure 7 which The authors are grateful to the referees for their helpful was consistent with previous reports that methylene green can comments on the manuscript. The authors thank Dr L. Wang be excluded out of DNA major groove (44,45). DNA CD sig- for his technical assistance on AFM experiments. This project nals were also changed in the presence of SWNTs (Figure 7). was supported by NSFC (20225102, 20331020, 20473084), When methylene green was out of DNA major groove, DNA and hundred people program from CAS. Funding to pay the CD spectrum was like the one for DNA–SWNTs in the Open Access publication charges for this article was provided absence of methylene green (Figure 3B), further supporting by the National Natural Science Foundation of China and that SWNTs bound to DNA in the major groove by replace- Chinese Academy of Sciences. ment of methylene green molecules. In combination with CD Conflict of interest statement. None declared. data, thermal denaturation, competitive binding assay and condensation results, SWNTs bound to the DNA major groove but not the minor groove, in agreement with REFERENCES SWNT–DNA simulation results which show that SWNTs 1. Iijima,S. (1991) Helical microtubules of graphitic carbon. 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Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B–A transition in solution

Li, Xi; Peng, Yinghua; Qu, Xiaogang
Nucleic Acids Research , Volume 34 (13) – Jan 1, 2006
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 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1093/nar/gkl513
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Published online August 2, 2006 3670–3676 Nucleic Acids Research, 2006, Vol. 34, No. 13 doi:10.1093/nar/gkl513 Carbon nanotubes selective destabilization of duplex and triplex DNA and inducing B–A transition in solution Xi Li, Yinghua Peng and Xiaogang Qu* Division of Biological Inorganic Chemistry, Key Laboratory of Rare Earth Chemistry and Physics, Graduate School of the Chinese Academy of Sciences, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China Received May 5, 2006; Revised June 6, 2006; Accepted July 5, 2006 single stranded DNA self-assembles into a helical structure ABSTRACT around individual carbon nanotubes. Since carbon nanotube– Single-walled carbon nanotubes (SWNTs) have DNA hybrids have different electrostatic properties that been considered as the leading candidate for nano- depend on the diameter of the nanotubes and electronic prop- device applications ranging from gene therapy erties, they can be separated and sorted using anion exchange and novel drug delivery to membrane separations. chromatography (11,12). Carbon nanotubes are able to con- The miniaturization of DNA-nanotube devices for dense double stranded plasmid DNA to varying degree and exhibit upregulation of marker gene expression over naked biological applications requires fully understanding DNA using a mammalian (human) cell line, a nanotube- DNA-nanotube interaction mechanism. We report based gene-delivery vector has been reported (16). In a recent here, for the first time, that DNA destabilization report, a piece of double-stranded DNA wrapped on the sur- and conformational transition induced by SWNTs face of a single-walled carbon nanotube can serve as sensors are sequence-dependent. Contrasting changes for in living cells (17) and the heart of the new optical detection SWNTs binding to poly[dGdC]:poly[dGdC] and system is based on the transition of DNA secondary structure poly[dAdT]:poly[dAdT] were observed. For GC from the native, right-handed ‘B’ form to the alternate, left- homopolymer, DNA melting temperature was handed ‘Z’ form which was modulated by metal ions. decreased 40 C by SWNTs but no change for Therefore, it is important and fundamental to understand AT-DNA. SWNTs can induce B–A transition for GC- the interaction mechanism of SWNT with double-stranded DNA but AT-DNA resisted the transition. Our circular DNA for nanodevice application. dichroism, competitive binding assay and triplex In this report, SWNTs DNA binding mode, binding preference and the impact on DNA stability and conformation destabilization studies provide direct evidence that were studied. Contrasting changes for SWNTs binding to SWNTs induce DNA B–A transition in solution and poly[dGdC]:poly[dGdC] and poly[dAdT]:poly[dAdT] were they bind to the DNA major groove with GC observed. We report here, for the first time, that DNA preference. condensation, destabilization and conformational transition induced by SWNTs are sequence-dependent. Our circular dichroism (CD), competitive binding assay and triplex INTRODUCTION destabilization studies provide direct evidence that SWNTs Single-walled carbon nanotubes (SWNTs) have been con- induce DNA B–A transition in solution and they bind to sidered as the leading candidate for nanodevice applications the DNA major groove with GC preference. The sequence- because of their one-dimensional electronic band structure, dependent condensation and B–A transition by SWNTs molecular size, biocompatibility, controllable property of shed light on the design of miniature of optical devices and conducting electrical current and reversible response to bio- label-free detection of specific genes. chemical reagents (1–6). These potential applications range from gene therapy and drug delivery to membrane separa- tions (4–16). Among the molecules that can non-covalently MATERIALS AND METHODS bind to the surface of SWNTs, DNA has been the research SWNTs (f ¼ 1.1nm, purity >90%) were purchased from focus (7–16), which adsorbs as a single-strand or double- Aldrich and purified as described previously by sonicating strand complexes. By screening a library of oligonucleotides, SWNTs in a 3:1 v/v solution of concentrated sulfuric acid previous reports have shown that a particular sequence of *To whom correspondence should be addressed. Tel: 86 431 526 2656; Fax: 86 431 5262656; Email: [email protected] 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2006, Vol. 34, No. 13 3671 (98%) and concentrated nitric acid (70%) for 24h at 35–40 C striking differences were observed among these DNA and washed with water, leaving an open hole in the tube side molecules (Figure 1D–F). SWNTs condense DNA depending and functionalized the open end of SWNTs with carboxyl on DNA GC content: GC homopolymer, GC-DNA was con- group to increase their solubility in aqueous solution (2). densed (23) as a network (Figure 1D). For calf thymus The stock solution of SWNTs (0.15 mg mL ) was obtained DNA (ct-DNA) and AT-DNA, they looked like forming by sonicating the SWNTs for 8 h in pH 7.0 aqueous solution. DNA-wrapped complexes and condensed slightly Calf thymus DNA (ct-DNA) was obtained from Sigma and (Figure 1E and F) showing that GC-DNA was more easily purified as described earlier (18). Poly[dGdC]:poly[dGdC] condensed than AT-DNA and ct-DNA. Premilat et al. have (GC-DNA), poly[dAdT]:poly[dAdT] (AT-DNA), polydA, measured the major groove width of GC-DNA (1.35 nm) and polydT were purchased from Pharmacia. The concentra- and AT-DNA (1.75 nm) through fiber X-ray diffraction tion of ct-DNA, GC-DNA and AT-DNA were determined by (26,27). SWNTs (f ¼ 1.1nm) we used were modified with ultraviolet absorbance measurements using the extinction carboxyl group. Carboxyl groups at the open end of 1 1 1 coefficient: e ¼ 12 824 M cm , e ¼ 16 800 M SWNTs greatly increased their water solubility and may 260 262 1 1 1 cm , e ¼ 13200 M cm , respectively (18). Triplex impact DNA binding to the modified nanotube surface. DNA (polydA:[polydT] ) was prepared as described previ- Based on SWNTs size, hydrophobic property and their ously (19–21). Ethidium bromide (EB), hoechst 33258 and improved solubility, SWNTs should not bind to DNA daunomycin (DM) were purchased from Sigma, methylene minor grooves due to the narrower groove width. Alternat- green was purchased from Aldrich and were used without fur- ively, SWNTs may bind to the major groove and would fit ther purification. Their concentrations were determined by better to GC-DNA major groove because AT-DNA major absorbance measurements using the extinction coefficient: groove is too wide for SWNTs binding. Hansma and Kankia 1 1 1 1 e ¼ 5600 M cm , e ¼ 42 000 M cm , have reported that condensation of DNA by metal ions is 480 338 1 1 e ¼ 11 500 M cm for EB, Hoechst 33 258 and DM, sequence dependent owing to the difference of GC and AT respectively (22). All the experiments were carried out in sequence in their extent of dehydration (23,28,29). GC-rich regions are being more heavily dehydrated than AT-rich Tris buffer (10 mM Tris, pH ¼ 7.1) unless stated otherwise. In sodium iodide fluorescence quenching experiments, the regions, this can be the reason why GC-DNA was easily con- ionic strength was kept constant. densed by SWNTs and this will be further discussed in our An AFM (Nanoscope IIIa, Digital Instruments, Santa Bar- CD studies. SWNTs, as gene-delivery vector (16), can take bara, CA) was used to image all DNAs in the presence or advantage to select highly GC-content gene for delivery absence of SWNTs. The sample solution was deposited and this needs to be verified in various gene expression onto a piece of freshly cleaved mica and rinsed with water systems. and dried before measurements (23). Tapping mode was used to acquire the images under ambient condition. CD SWNTs selective destabilization of DNA spectra were measured at 20 C on a JASCO J-810 spectro- polarimeter with a computer-controlled water bath (24). Figure 2 shows DNA UV melting profiles in the absence or The optical chamber of CD spectrometer was deoxygenated presence of SWNTs. Contrasting changes for SWNTs binding with dry purified nitrogen (99.99%) for 45 min before use to GC-DNA and AT-DNA were observed. It is obvious that and kept the nitrogen atmosphere during experiments. GC-DNA and ct-DNA became unstable in the presence of Three scans were accumulated and automatically averaged. SWNTs. Melting temperature T decreased 40 C for GC- Absorbance measurements and melting experiments were DNA when SWNTs at 25 mg/ml (Figure 2A). The absorption made on a Cary 300 UV/Vis spectrophotometer, equipped after 80 C decreased showing the strong interaction of single- with a Peltier temperature control accessory (25). All UV/ strand DNA with SWNTs (11,12). Since the melting temper- Vis spectra were measured in 1.0 cm path length quartz ature of GC-DNA in the presence of 15 mgml or higher SWNTs concnentration was much lower than 60 C, it cuvettes with the same concentration of SWNTs aqueous seems unlikely that the destabilization was due to pH change solution as the reference. Absorbance changes at 260 nm with temperature. On the contrary to GC-DNA and ct-DNA, versus temperature were collected at a heating rate of SWNTs did not influence AT-DNA stability even at high 0.5 C min for DNA melting experiments. Primary data concentration of SWNTs (Figure 2C). At 20 mM NaCl, were transferred to the graphics program Origin for plotting similar trend for SWNTs bound to DNA was observed and analysis. Fluorescence experiments were carried out on a JASCO FP-6500 spectrofluorometer at 20 C (24). (data not shown). We also studied SWNTs bound to triplex DNA at high salt conditions which will be addressed in triplex destabilization section. These results showed that the binding preference of SWNTs was: GC-DNA>ct-DNA>AT- RESULTS AND DISCUSSION DNA, consistent with the AFM and CD results which will SWNT inducing sequence-dependent be addressed next. DNA condensation AFM studies showed that DNA condensed when they bound SWNTs making DNA B–A transition in solution to carboxyl-modified SWNTs (2) and the condensation was dependent on DNA composition. In the absence of SWNTs, As DNA bound to carboxyl-modified SWNTs, various inter- linear GC-DNA, AT-DNA and natural DNA, purified calf actions of DNA bases and backbone with SWNTs, such as hydrophobic interactions, van der Waals, electrostatic inter- thymus DNA (ct-DNA) were observed (Figure 1A–C). actions, can take place (15). The strong interactions between When they bound with carboxyl-modified SWNTs, however, 3672 Nucleic Acids Research, 2006, Vol. 34, No. 13 Figure 1. DNA AFM images in the absence or presence of SWNTs: (A) GC-DNA alone; (B) ct-DNA alone; (C) AT-DNA alone; (D) GC-DNA + 40 mgml 1 1 1 SWNTs; (E) ct-DNA + 40 mgml SWNTs; (F) AT-DNA + 40 mgml SWNTs. The DNA concentration used in all experiments was 19.5 mgml . All the AFM images are captured on freshly cleaved mica. The image for (A–C) is 500 nm · 500 nm and the same scale bar. The image for (D–F) is 1 mm · 1 mm and with the same scale bar. SWNTs and DNA can disturb DNA hydration layer, even make DNA B–A transition in solution, consistent with DNA structure (22,29). previous molecular dynamics simulation results which show CD spectra showed these DNA molecules were in B-form B–A transition when DNA encapsulated in carbon nanotube (Figure 3) with a positive band near 270 nm and a negative or on gold surface (7,9). The induced B to A transition was band near 250 nm in the absence of SWNTs (18,24). Upon due to SWNTs bound to the major groove resulting in deep- addition of SWNTs (Figure 3A), the canonical B form of ening and narrowing the major groove while widening the GC-DNA altered with a positive band near 258 nm and a minor groove, which was coincident with the previous simu- negative band near 242 nm indicating that B–A transition lation results (33). (28–33) occurred. The transition was cooperative and the Calf thymus DNA (ct-DNA, 42% GC and 58%AT) was transition midpoint was at 10 mg/ml SWNTs (Figure 4). CD induced to A-form by SWNTs but not as easily as GC-DNA spectroscopy provided the direct evidence that SWNTs could while AT-DNA persisted in B-form (Figure 3B and C), Nucleic Acids Research, 2006, Vol. 34, No. 13 3673 1.0 A-Form 0.5 −1 +25 µg ml DNA alone SWNTs 0.0 30 45 60 75 90 −2 1.0 B-Form −4 CD258nm 0.5 CD252nm −6 0.0 −8 30 40 50 60 70 80 0 5 10 15 20 25 1.0 Conc (SWNT), µg/ml Figure 4. Plot of CD intensity versus concentration of SWNTs. CD intensity 0.5 at 258 nm (solid circles) and at 252 nm (open circles). The data were adopted from Figure 3A. 0.0 showing that B–A transition was dependent on the G-C con- 30 40 50 60 70 80 tent of the DNA helix (28–33). Ivanov and Krylov (34) have Temperature (°C) reviewed the cooperative character of B–A transition and determined the cooperative width of B–A transition with Figure 2. UV melting profiles of DNA: (A) poly[dGdC]:poly[dGdC], three different methods, and confirm that the cooperative (B) ct-DNA, (C) poly[dAdT]:poly[dAdT] in the absence or presence of width of B–A transition for DNA with mixed sequence is SWNTs. From right to left: 0, 1, 5, 10, 15, 20, 25 mgml SWNTs in pH ¼ 7 in the range of 10–30 bp. Since GC-DNA was more easily solution. Normalized absorption changes at 260 nm were plotted against temperature. The data were collected at a heating rate of 0.5 C min . Details condensed by SWNTs, the width of the transition for GC see Materials and Methods. homopolymer should be lower than that for the DNA with mixed sequence (31), such as ct-DNA. Since the water activ- ity is an apparent driving force for B–A transition and the 5 A-Form water activity (31) of GC-rich region (81.2) is lower than AT-rich region (81.5), GC-DNA undergoes the B to A trans- −5 ition most easily, whereas AT-DNA resists the B to A trans- B-Form ition. Previous studies show that GC homopolymer have a −10 stronger tendency for aggregation than AT homopolymer, −15 and GC homopolymer can undergo B–Z–A transition by reducing water activity (35). Under the usual experimental 10 conditions for B–A transition, GC homopolymer will aggreg- ate (36) while AT homopolymer would remain in the B-form even at conditions which normally favors the A-form (36,37). These results further support that GC homopolymer was more easily condensed by SWNTs than AT homopolymer as shown −5 in our AFM studies. A-DNA is biologically relevant and has 11 bp per helical turn, base pairs are tilted to 20 C with respect to the helical axis (38), the grooves are not as deep as those in B-DNA, the 0 0 sugar pucker is C3 endo compared with C2 endo for B-DNA. Like B–Z DNA transition (17,18,24,39,40), the transition −3 from the B-DNA double helix to the A-form is essential for biological function (28–33), as shown by the existence of 200 220 240 260 280 300 320 the A-form in many protein–DNA complexes, increasing the Wavelength (nm) fidelity of DNA and RNA synthesis and protection from DNA damage. Figure 3. CD spectra of DNA. (A) poly[dGdC]:poly[dGdC] (B) ct-DNA (C) poly[dAdT]:poly[dAdT] in the absence (black) or presence of SWNTs: Fluorescence competitive binding assay and triplex SWNTs 1 (red), 5 (green), 10 (blue), 15 (cyan), 20 (magenta) and 25 mgml (yellow) in Tris buffer (pH ¼ 7.1) at 20 C. CD spectra were measured on a DNA destabilization by SWNTs JASCO J-810 spectropolarimeter with a computer-controlled water bath as It is well known that EB and DM can intercalate into DNA described in Materials and Methods. Three scans were accumulated and automatically averaged. through the minor groove and Hoechst 33 258 is a classical Normalized A Ellipticity (mDegree) CD Intensity 3674 Nucleic Acids Research, 2006, Vol. 34, No. 13 A 30 A 500 550 600 650 700 400 450 500 550 600 650 C 0 50 100 150 200 Concentration of Iodide (mM) Figure 6. Plot of fluorescence intensity of fluorephore-DNA (squares) or fluorephore-DNA–SWNTs (circles) versus NaI concentration. The ionic strength was kept constant: (A)10 mM EB (Excitation wavelength: 480 nm, 500 550 600 650 700 Emission wavelength: 590 nm Slit: 5 nm); (B)10 mM Hoechst 33 258 (Excitation wavelength: 355 nm, Emission wavelength: 450 nm Slit: 3 nm); Wavelength (nm) (C)10 mM DM (Excitation wavelength: 480 nm, Emission wavelength: 565 nm Slit: 10 nm). Experimental details described in the experimental section. 300 D 590nm 450nm 10 565nm −3 −2 −10 −10 Log (Conc(SWNT), µg/ml) 300 400 500 600 700 Wavelength (nm) Figure 5. Fluorescence emission spectra of (A)1 mM EB (Excitation wavelength: 480 nm, Slit: 10 nm); (B)1 mM Hoechst 33 258 (Excitation wavelength: 355 nm, Slit: 3 nm); (C)1 mM DM (Excitation wavelength: 480 Figure 7. Loss of CD signal from calf thymus DNA-methylene green nm, Slit: 10 nm): 1mM fluorophore (EB, Hoechst 33 258, DM) alone (black complex upon SWNT–DNA association. Methylene green (black); calf curves), 1 mM fluorophore + 30 mgml CT-DNA (open circles), 1 mM thymus DNA (red); calf thymus DNA-methylene green (green); calf thymus 1 1 fluorophore + 30 mgml CT-DNA + 1 mgml SWNTs (upper triangles). DNA-methylene green after association with SWNTs at binding ratio r ¼ 0.2 (D) Fluorescence intensity as a function of concentration of SWNTs: 1 mM (blue). Calf thymus DNA was 50 mM in bp. CD spectra were measured at 1 1 EB + 30 mgml CT-DNA (squares), 1 mM Hoechst 33 258 + 30 mgml 20 C on a JASCO J-810 spectropolarimeter with a computer-controlled water CT-DNA (circles), 1 mMDM + 30 mgml CT-DNA (triangles). The bath as described in the experimental section. Three scans were accumulated experiments were carried out in Tris buffer (pH ¼ 7.1) at 20 C. and automatically averaged. DNA minor groove binder. When bound to DNA, the fluores- not competitively bind to the same sites. To further identify cence of EB or Hoechst is greatly enhanced, and DM fluores- their different binding sites, we carried out NaI quenching cence is strongly quenched. With this in mind, if SWNTs experiments. Iodide ions cannot quench the fluorescence of competitively bind to the same sites of DNA as EB, Hoechst these dye molecules when they are bound to DNA (43). If and DM, the fluorescence of EB and Hoechst would decrease SWNTs could replace these molecules, their fluorescence and the fluorescence of DM would increase because the should be quenched. Figure 6 showed that the fluorescence strong binding of SWNTs with DNA should exclude was not quenched by iodide at all. However, we found that these DNA binders out of their binding sites. The fluores- SWNTs could exclude methylene green, a proven DNA cence competitive binding assay has been widely used major groove binder (44,45), out of DNA. Figure 7 showed to establish DNA binding mode (41–43). As shown CD spectral changes in the presence of SWNTs. For methyl- in Figure 5A–C, their fluorescence hardly changed, even ene green, there was no CD signal in our experimental condi- titrated by SWNTs (Figure 5D), showing that SWNTs do tions. When methylene green bound to DNA, three induced Fluorescence Intensity (a.u) Fluorescence Intensity (a.u) I I I 565nm 450nm 590nm Ellipticity (mDegree) Nucleic Acids Research, 2006, Vol. 34, No. 13 3675 1.0 therapeutic agents, and because of the possible relevance of H-DNA structures in biology system (19–21). However, 0.8 SWNTs selective destabilization of triplex DNA has not been reported. Based on previous computer simulation results 0.6 of SWNTs binding to duplex DNA in the major groove (14) 0.4 and our melting, CD, and competitive binding data, SWNTs probably bound to duplex DNA major groove and had GC 0.2 preference. 0.0 30 40 50 60 70 80 90 CONCLUSIONS Temperature (°C) SWNTs can cause sequence-dependent DNA condensation Figure 8. UV melting profiles of triplex DNA polydA (polydT) in the and strongly destabilize GC-DNA. Contrasting changes for 1 1 absence (black) or presence of SWNTs: SWNTs 1 mgml (red); 2 mgml 1 1 SWNTs binding to GC-DNA and AT-DNA were observed. (green); 5 mgml (blue); 10 mgml (cyan) in Tris buffer (10 mM Tris, Our CD, competitive binding and triplex destabilization stud- 200 mM NaCl, pH ¼ 7.1). Normalized absorption changes at 260 nm were plotted against temperature. The data were collected at a heating rate of ies provide direct evidence that SWNTs induce DNA B–A 0.5 C min . transition in solution and they bind to the DNA major groove with GC preference. CD signals characteristic of bound methylene green around 310 nm, 430 nm, 650 nm were observed (44,45). With addi- ACKNOWLEDGEMENTS tion of SWNTs, the induced CD intensity was decreased and even disappeared, typical data was shown in Figure 7 which The authors are grateful to the referees for their helpful was consistent with previous reports that methylene green can comments on the manuscript. The authors thank Dr L. Wang be excluded out of DNA major groove (44,45). DNA CD sig- for his technical assistance on AFM experiments. This project nals were also changed in the presence of SWNTs (Figure 7). was supported by NSFC (20225102, 20331020, 20473084), When methylene green was out of DNA major groove, DNA and hundred people program from CAS. Funding to pay the CD spectrum was like the one for DNA–SWNTs in the Open Access publication charges for this article was provided absence of methylene green (Figure 3B), further supporting by the National Natural Science Foundation of China and that SWNTs bound to DNA in the major groove by replace- Chinese Academy of Sciences. ment of methylene green molecules. In combination with CD Conflict of interest statement. None declared. data, thermal denaturation, competitive binding assay and condensation results, SWNTs bound to the DNA major groove but not the minor groove, in agreement with REFERENCES SWNT–DNA simulation results which show that SWNTs 1. Iijima,S. (1991) Helical microtubules of graphitic carbon. 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Published: Jan 1, 2006

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