Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes

Accepted Manuscript Title: Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes Author: Daisuke Nii Takuya Hayashida Yuuki Yamaguchi Shukuko Ikawa Takehiko Shibata Kazuo Umemura PII: DOI: Reference:

S0927-7765(14)00293-8 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.06.008 COLSUB 6454

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

6-2-2014 23-5-2014 3-6-2014

Please cite this article as: D. Nii, T. Hayashida, Y. Yamaguchi, S. Ikawa, T. Shibata, K. Umemura, Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.06.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes

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Daisuke Nii a, Takuya Hayashida a, Yuuki Yamaguchi b,c, Shukuko Ikawa b, Takehiko Shibata b, Kazuo

Biophysics Section, Department of Physics, Graduate School of Science, Tokyo University of

Science, 1-3 Kagurazaka, Shinjuku, Tokyo 1628601, Japan b

Cellular and Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198,

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Japan

Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku,

Yokohama, Kanagawa, 230-0045, Japan

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Prof. Kazuo Umemura

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*Corresponding author:

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Umemura a*

Biophysics Section, Department of Physics, Graduate School of Science, Tokyo University of

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Science, 1-3 Kagurazaka, Shinjuku, Tokyo, Japan, 1628601 Tel:

Fax: +81-3-5228-8228

E-mail address: [email protected]

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Abstract Single-stranded DNA-binding (SSB) proteins were treated with hybrids of DNA and single-walled

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carbon nanotubes (SWNTs) to examine the biological function of the DNA molecules adsorbed on the SWNT surface. When single-stranded DNA (ssDNA) was used for the hybridization, significant

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binding of the SSB molecules to the ssDNA-SWNT hybrids was observed by using atomic force

microscopy (AFM) and agarose gel electrophoresis. When double-stranded DNA (dsDNA) was used,

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the SSB molecules did not bind to the dsDNA-SWNT hybrids in most of the conditions that we evaluated. A specifically modified electrophoresis procedure was used to monitor the locations of the

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DNA, SSB, and SWNT molecules. Our results clearly showed that ssDNA/dsDNA molecules on the

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SWNT surfaces retained their single-stranded/double-stranded structures.

Keywords:

single-stranded DNA binding protein; carbon nanotube; atomic force microscopy; agarose gel

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electrophoresis; molecular recognition

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1. Introduction Techniques for the hybridization of DNA and single-walled carbon nanotubes (SWNTs) were

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first proposed by two independent research groups in 2003 [1-2]. When SWNT powder was mixed with DNA molecules in solution and sonicated, the DNA molecules were adsorbed onto debundled

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SWNT surfaces. Because the resulting DNA-SWNT hybrids were water soluble, the hybridization techniques were originally recognized as solubilization techniques for insoluble SWNT powder.

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Since the establishment of DNA-SWNT hybridization methods, various biological applications using the DNA-SWNT hybrids have been proposed over the last ten years [3-9].

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One promising approach for the biological application of DNA-SWNT hybrids involves the combination of protein molecules with the DNA-SWNT hybrids since the addition of protein

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molecules would provide several rich functions. Protein molecules have typically been attached to DNA-SWNT hybrids via covalent bonding by chemical modification of the 5′- or 3′termini of

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the DNA molecules attached to the SWNT surfaces. For example, in previous studies, one end of a DNA molecule was modified with biotin, to which streptavidin molecules were then attached

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[10-12]. In those studies, even though whole DNA molecules were employed, only the termini of the

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DNA molecules were used for binding. In another approach, researchers attached proteins onto bare SWNT surfaces or chemically functionalized the SWNT surfaces without using DNA molecules [13-19].

Although various protein molecules have previously been used with DNA-SWNT hybrids or

bare SWNT molecules, there is currently no example that demonstrates the reaction of DNA-binding proteins with DNA-SWNT hybrids based on the biological recognition between the protein and DNA molecules. Indeed, our literature review found very few related papers. Keren et al. developed a field-effect transistor (FET) using RecA, DNA, and SWNT molecules [20-22]. In this case, RecA proteins were first treated with the DNA molecules, and the RecA-DNA hybrids were then attached to the SWNT molecules, which was not a direct reaction between RecA and the DNA-SWNT hybrids. Kerman et al. clearly demonstrated the selective binding of single-stranded DNA-binding (SSB) proteins onto SWNT electrodes modified with single-stranded DNA (ssDNA) or 3

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double-stranded DNA (dsDNA) [23]. In their study, the SWNT electrode surfaces were coated with ssDNA or dsDNA molecules, and the SSB proteins were then treated with the functionalized electrode surfaces. Under these conditions, the SSB proteins likely interacted with the free DNA

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molecules on the SWNT electrode, and not with the DNA-SWNT hybrids. While DNA-binding

DNA-SWNT hybrids was not the primary focus of the authors’ work.

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proteins were employed in these studies, the reaction of DNA-binding proteins with the

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In the current study, we focused on reactions of SSB proteins with DNA-SWNT hybrids. It is known that SSB proteins selectively bind to ssDNA, but not to dsDNA molecules [24]. Although the biological applications of the DNA-SWNT hybrids have been previously studied, the structures and

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properties of the DNA molecules on SWNT surfaces have not been elucidated. Specifically, while researches employing dsDNA-SWNT hybrids have recently increased, structures of the

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dsDNA-SWNT hybrids have not been well investigated [2, 25-32]. While some reports suggest that dsDNA molecules change to ssDNA molecules when the dsDNA molecules bind to the SWNT

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surfaces, extensive experimental evidence is lacking [33]. Here, we wanted to verify that SSB

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proteins selectively bind only to the ssDNA-SWNT hybrids, and not to the dsDNA-SWNT hybrids. These results would mean that ssDNA and dsDNA molecules in the DNA-SWNT hybrids still

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possess their biological function as DNA molecules. However, if the SSB proteins were not able to distinguish between the ssDNA and dsDNA molecules in the DNA-SWNT hybrids, it would suggest that the structure of the DNA molecules was deformed during hybridization with the SWNTs. Electrophoresis and atomic force microscopy (AFM) were used for characterization of the

hybrid molecules. Electrophoresis techniques have previously been employed to characterize both DNA-SWNT hybrids and protein-SWNT hybrids [34-41]. For example, Huang et al. examined hybrids of bovine serum albumin (BSA) and SWNT using acrylamide gel electrophoresis [34]. In this case, most of the SWNT remained in the loading wells, even after the electrophoresis, and therefore, the BSA proteins that were attached to the SWNT also remained in the loading wells. Previous characterization of DNA-SWNT hybrids was performed using agarose gels stained with ethidium bromide (EtBr) and then observed under UV and visible light. Under UV and visible light,

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the locations of DNA and SWNT molecules were identified, respectively [35, 36, 39, 41]. There are no previous reports examining DNA-SWNT hybrids with proteins. We, therefore, attempted to identify the locations of DNA, SSB, and SWNT molecules by specifically modifying an

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electrophoresis procedure.

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2. Materials and methods

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2.1. Materials

SSB proteins, ssDNA consisting of 30-mer of thymine (dT30), and dsDNA (Salmon testes, D1626) were purchased from Bio Academia Inc. (Osaka, Japan), Life Technologies Japan Ltd.

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(Tokyo, Japan), and Sigma-Aldrich Co. (St. Louis, MO, respectively. The DNA powders were dissolved in 20 mM Tris-HCl buffer solution (pH 7.5). Because the dsDNA does not completely

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dissolve owing to its nature, the dsDNA sample was sonicated using a bath-type sonicator (LEO-80, Steady Ultrasonic Sdn. Bhd., Selangor, Malaysia) for 30 min at 80 W. The concentration of the

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sonicated dsDNA solution was estimated using UV-Vis spectroscopy. HiPco SWNT powder (Super

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Purified HiPCO SWNTs) was purchased from Unidym, Inc. (Sunnyvale, CA).

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2.2. Preparation of the DNA-SWNT hybrids

DNA-SWNT hybrids were prepared as follows: 0.5 mg of SWNT powder was mixed with 1

mL of ssDNA or the dsDNA solution (containing 0.5 mg of ssDNA or dsDNA). The mixture was sonicated with a probe-type sonicator (VCX 130, Sonics & Materials, Inc., CT, USA) in an ice-water bath for 90 min with a 2-mm diameter tip at 60% amplitude vibration [32]. Subsequently, the sample was centrifuged for 3 h at 17,360 × g, (MX-150, Tomy Seiko Co., Ltd., Tokyo, Japan) The supernatant was collected after centrifugation, and it was used for all of the following experiments.

2.3. Binding of the SSB proteins to DNA-SWNT hybrids For the binding of SSB proteins, 8 μL of the ssDNA/dsDNA-SWNT hybrid solution was mixed with 1 μL of the SSB solution and with 1 μL of the buffer solution containing 200 mM NaCl

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and 100 mM MgCl2 [42,43]. The final SSB concentrations after mixing were 0, 25, 50, 75, 100, and 500 μg/mL. The final concentration of DNA and SWNT was 400 μg/mL in the solution, assuming that no loss occurred following centrifugation. The SSB and ssDNA/dsDNA-SWNT hybrid mixtures

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were incubated at 37°C for 10 or 30 min. For AFM and transmission electron microscopy (TEM), samples were prepared at a lower concentration. In this case, the final concentration of DNA and

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SWNT was 2 μg/mL, and the SSB concentrations were 0 and 2 μg/mL. For electrophoresis samples,

that of the electrophoresis and then diluted after the reaction.

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2.4. Agarose gel electrophoresis

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some were prepared at a higher concentration, and others were prepared at the same concentration as

Agarose gel electrophoresis (Mupid-2plus, ADVANCE CO., LTD., Tokyo, Japan) was carried

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out using a 0.8% gel (Agarose S, Wako Pure Chemical Industries, Ltd., Osaka, Japan). A volume of 10 μL of the aforementioned mixture of SSB, ssDNA/dsDNA, and SWNT was mixed with 2.5 μL of

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glycerol, after which 10 μL of the sample was loaded. A volume of 1 μL of Coomassie Brilliant Blue

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solution (0.25% CBB R-250, 5% methanol, 7.5% acetic acid) was also added to the sample mixture when protein bands were monitored. In this case, the gel was observed under visible light after the

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electrophoresis without staining EtBr. The electrophoresis was carried out at 50 V for 30 min in TAE buffer solution. Electrophoresis examination was repeated more than three times to verify reproducibility.

To monitor the position of the DNA molecules, the gel was stained with 1.0 µg/mL EtBr for 30

min, and then observed using a UV transilluminator (Mupid scope WD, ADVANCE CO. Ltd., Tokyo, Japan). For monitoring the SWNT locations, the same gel was observed under visible light (5000 K, Light Viewer 5700, HAKUBA PHOTO INDUSTRY CO., LTD, Tokyo, Japan). When CBB R-250 was added to the sample prior to loading of the gel, the gel was observed under visible light without additional staining.

2.5. Atomic force microscopy

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AFM evaluation was performed using the AC-AFM mode (MFP-3D microscope, Asylum Research, CA, USA) with a silicon cantilever (OMCL-AC240TS-R3, Olympus Co, Tokyo, Japan) in air. A volume of 10 µL of each sample was deposited onto a mica surface pretreated with 0.01% APS

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solution. After incubating for 10 min at room temperature, the samples were rinsed with 1 mL of

pure water and dried. The height of the DNA-SWNT hybrids and the SSB-DNA-SWNT hybrids was

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measured by cross-sectional analysis of the obtained AFM images. The average height values of the

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hybrids were calculated from 300 cross-sections of 100 molecules (3 positions for one hybrid) for each sample.

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2.6. Transmission electron microscopy

TEM observation was carried out using a JEM-1230 (JEOL Co., Tokyo, Japan) microscope. A

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metal grid was pre-coated with paradione and carbon, and treated by high-voltage glow discharge (JFC-1100E ION SPUTTER, JEOL Co., Tokyo, Japan). A volume of 3 µL of each sample solution

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3. Results and discussion

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of accelerating voltage.

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was deposited on the grid and then stained with uranyl acetate. The images were obtained at 80 kV

Our experiments were conducted according to the schematic shown in Fig. 1. Prior to the SSB

reaction, the ssDNA/dsDNA and SWNT mixture was sonicated to prepare the ssDNA/dsDNA-SWNT hybrids. The SSB molecules were then treated with the DNA-SWNT hybrids in a standard buffer solution containing NaCl and MgCl2 [42, 43]. Although preparation and characterization of the DNA-SWNT hybrids has been widely studied, the function of DNA molecules adsorbed on to the SWNT surfaces is not yet entirely clear. Reaction of the DNA-SWNT hybrids with SSB proteins may provide valuable information regarding the structure and function of the DNA on the SWNT surfaces since SSB proteins selectively bind to the base side of ssDNA. AFM images of ssDNA/dsDNA-SWNT hybrids incubated with and without SSB proteins for 10 min at 37°C are shown in Fig. 2. When SSB proteins were treated with ssDNA-SWNT hybrids

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(Fig. 2(b)), thick, rod-like structures were observed as opposed to the typical ssDNA-SWNT hybrids (Fig. 2(a)), indicating the creation of SSB-ssDNA-SWNT hybrids. Although free ssDNA molecules were probably present in the sample, the ssDNA (dT30) was likely too small to observe. To validate

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this, the height of the hybrids was measured from 300 cross-sections of 100 molecules in the AFM images (Fig. 2(c)). For the ssDNA-SWNT without SSB proteins, the height distribution was from

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0.85 nm to 3.74 nm. When SSB proteins were treated with the hybrids, a wide-ranged height

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distribution was obtained (the red rectangle in Fig. 2(c)). The data clearly indicated that SSB proteins formed bonds with the ssDNA-SWNT surfaces, which is why the ssDNA molecules on the SWNT surfaces were recognized as “ssDNA” by the SSB molecules. The minimum height of the

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SSB-ssDNA-SWNT was 0.68 nm, suggesting that ssDNA-SWNT hybrids without SSB were also present. In other words, the wide distribution indicates heterogeneous binding of the SSB proteins,

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which may suggest the cooperative binding of SSB molecules. The average height for the SSB-ssDNA-SWNT and ssDNA-SWNT was 1.5 ± 0.4 and 5.0 ± 1.6 nm, respectively. The large

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standard error quantitatively revealed the wide distribution of SSB-ssDNA-SWNT hybrids. Similar

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samples were also verified with TEM observation (Fig. S1). Fig. 2(d) and 2(e) show the dsDNA-SWNT hybrids without and with SSB proteins,

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respectively. In this case, the images and the height distributions revealed no differences between the two samples. It appeared that SSB proteins were not attached to the dsDNA-SWNT surfaces, suggesting that the dsDNA molecules on the SWNT surface were recognized as “dsDNA” by the SSB proteins. Although dsDNA was sonicated during preparation of the dsDNA-SWNT hybrids, the dsDNA structures likely remained unchanged. Free dsDNA molecules were also sometimes observed in AFM images, and were easily distinguished from dsDNA-SWNT hybrids because the free dsDNA molecules did not exhibit a rod-like shape. The average heights of the hybrids were 1.7 ± 0.6 and 1.8 ± 0.7 nm without and with the SSB molecules, respectively. In addition, many circular objects, 2–3 nm in height, were observed in Fig. 2(e). Because the molecular weight of the SSB proteins (tetramer) is 76,500 Da, based on the size of the observed circular objects, it is reasonable that they may be considered to be free SSB molecules. When similar samples were incubated for 30 min at

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37°C, no significant differences was observed in AFM images (Fig. S2). To characterize the adsorption of SSB molecules onto the DNA-SWNT surface in more detail, we performed agarose gel electrophoresis (Fig. 3 and 4). For Fig. 3, the gel was stained with EtBr

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solution after the electrophoresis, and was observed under UV light to estimate the location of the

DNA molecules. The same gel was then observed under visible light to determine the location of the

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SWNTs. The incubation periods were 10 and 30 min in Fig. 3(a) and 3(b), respectively.

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The images obtained under visible light are shown in the top panel of Fig. 3, where the black bands indicate the location of SWNT. Because the insoluble and bare SWNT molecules must be removed by centrifugation during the sample preparation, these bands should represent only

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ssDNA-SWNT hybrids and not the bare SWNT. The apparent smearing features suggest variation of the SWNT length/width or the heterogeneity of DNA adsorption. In Fig. 3(a), lane 1 shows the

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initial position of the ssDNA-SWNT without the added SSB proteins. When ssDNA-SWNT hybrids were treated with SSB proteins (lanes 2–6), a clear upward shift of the ssDNA-SWNT band was

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observed according to the amount of added SSB proteins. These data indicate that the SSB proteins

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formed bonds with the ssDNA-SWNT molecules.

Lane 7 in Fig. 3(a) shows a band of dsDNA-SWNT. The initial position of the dsDNA-SWNT

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band was higher than that of the ssDNA-SWNT. Because the same SWNT powder was used in all of the experiments, the difference in the initial position was a result of the difference between the ssDNA and dsDNA. In the case of the dsDNA-SWNT, changes in the locations of the bands were not observed until a concentration of 75 μg/mL of SSB protein (lane 10) was used. When the SSB concentration was increased to 500 μg/mL, an upward shift of the SWNT location was evident (lane 12). The data indicated that the only when the concentration of SSB proteins was over 75 μg/mL did they bind to the dsDNA-SWNT hybrids, and that non-specific binding may have occurred in samples with the higher SSB concentrations. When similar samples were incubated for 30 min, differences that were more significant were observed (Fig. 3(b)). In the case of the ssDNA-SWNT, even at the lowest concentration of SSB proteins, the ssDNA-SWNT band was stopped at the loading well, suggesting that the molecular

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weight of the ssDNA-SWNT was drastically increased by binding of the SSB proteins. On the other hand, in the case of the dsDNA-SWNT, such a phenomenon was not observed, even at the highest SSB concentration (500 μg/mL, lane 12 in Fig. 3(b)). Based on these data, the selective binding of

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SSB proteins to the ssDNA-SWNT was clearly indicated.

Images obtained under UV light following EtBr staining are shown at the bottom panel of Fig.

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3. Bands of ssDNA (dT30), unfortunately, could not be observed. This was likely because the small

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molecules escaped from the gel during the electrophoresis. Although bright dsDNA bands were observed, their location was different from that of the dsDNA-SWNT observed in the top panel in Fig. 3. The dsDNA molecules seen in bottom panel in Fig. 3 may be free DNA molecules that were

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not attached to the SWNT. Bands of the DNA molecules were divided into two bands in lanes 11 and 12 in both Fig. 3(a) and (b). Thus, non-specific binding of the SSB proteins appeared in both the

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dsDNA-SWNT and the free dsDNA at high SSB concentrations (more than 100 μg/mL). Additional electrophoresis experiments were performed to evaluate the effects of sonication on dsDNA

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molecules (Fig. S3).

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Finally, we attempted to monitor the location of the SSB proteins with a modified agarose gel electrophoresis procedure. CBB staining is one of the most popular methods for monitoring protein

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location in a gel. However, when we applied the CBB staining method for our samples using the agarose gel electrophoresis, the agarose gel itself was stained by the CBB, rendering it impossible to observe protein bands (data not shown). It has been shown that if acrylamide gel electrophoresis is employed, the SWNT molecules are retained by the stacking gel, even without protein binding [34, 41]. For our samples, the CBB solution was mixed in the samples before electrophoresis and then observed with agarose gel electrophoresis (Fig. 4). With this method, free CBB molecules adsorbed to the loading well; therefore, analysis of bands at the loading wells was unnecessary (Fig. S3). However, some of the CBB molecules did migrate after the electrophoresis. Although the mechanism of this phenomenon is not clear, the migration of the CBB molecules can be used for characterization of the DNA-SWNT hybrids with SSB molecules. For example, in Fig. 4(a), smeared bands were observed in lanes 1 and 2. The SSB concentration in lanes 1 and 2 was 0 and 25 μg/mL,

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respectively. When the SSB concentration was increased, the smeared band gradually disappeared in the case of the ssDNA-SWNT hybrid samples (lanes 3–6 in Fig. 4(a) and lanes 2–6 in Fig. 4 (b)). On the other hand, in the case of the dsDNA-SWNT samples, the smeared bands did not disappear even

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when the SSB concentration was increased (lanes 8–12 both in Fig. 4(a) and 4(b)). Although the

mechanism of this phenomenon was not clear, the data supported the idea of selective binding of the

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SSB proteins only to the ssDNA-SWNT surfaces. It is likely that some of the CBB molecules were

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attached to the SSB molecules, and the resulting SSB-CBB complexes could bind to the ssDNA-SWNT hybrids.

Our results clearly revealed the specific binding of SSB proteins to ssDNA-SWNT hybrids

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and not to the dsDNA-SWNT hybrids. While further experiments are still necessary to fully conclude the binding mechanisms, based on this work, we are able to discuss possible ideas for SSB

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binding. If SWNT behaved in the same manner as a complementary ssDNA, the ssDNA-SWNT surface should be similar to that of a dsDNA. In this case, SSB would likely be unable to recognize

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the hybrid as an ssDNA molecule. Therefore, our results suggest that the base sides of ssDNA

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molecules were not completely situated close together on the SWNT surface. In the theoretical model of ssDNA-SWNT structures previously proposed by Zheng et al., the bases of ssDNA

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molecules are partially exposed outward [1]. Our data do not contradict with this theoretical model.

4. Conclusions

We have discovered that SSB proteins form complexes with ssDNA-SWNT hybrids. The

molecular recognition function of the SSB protein molecules has been previously studied for DNA molecules on SWNT surfaces. However, very little information was available on the hybridization of dsDNA and SWNTs. Our data suggested that dsDNA molecules preserve their double-stranded structures on the SWNT surfaces. We have thus demonstrated the potential of various applications based on the complexes of different DNA-binding proteins with DNA-SWNT hybrids, by showing that DNA-binding protein molecules are not damaged by sonication during sample preparation.

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Acknowledgements

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The authors thank Prof. T. Tomo for valuable advice for electrophoresis experiments. This work was

supported by a Grant-in-Aid for Scientific Research (23540479) of the Japan Society for the

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Promotion of Science (JSPS). This work was also supported by the Division of Nanocarbon

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Research at the Research Institute for Science & Technology (RIST) at the Tokyo University of

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Science.

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[37] R.Wang, C. Mikoryak, E. Chen, S. Li, P. Pantano, R.K. Draper, Anal Chem. 81 (2009) 2944.

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[38] M. Parthasarathy, J. Debgupta, B. Kakade, A.A. Ansary, M. Islam Khan, V.K. Pillai, Anal

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Biochem. 409 (2011) 230.

[39] I.I. Shakhmaeva, E.R. Bulatov, O.V. Bondar, D.V. Saifullina, M. Culha, A.A. Rizvanov, T.I. Abdullin, J Biotechnol. 152 (2011) 102.

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[40] M. Gunavadhi, L.A. Maria, V.N. Chamundeswari, M. Parthasarath, Electrophoresis. 33 (2012) 1271.

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[41] D.Nii, T,Hayashida, K. Umemura, Colloids Surf. B 106 (2013) 234. [42] L. Hamon, D. Pastré, P. Dupaigne, C. Le Breton, E. Le Cam, O. Piétrement, Nucleic Acids Res.

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35 (2007) e58.

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1500.

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[43] L.S. Shlyakhtenko, A.Y. Lushnikov, A. Miyagi, Y.L. Lyubchenko, Biochemistry. 51 (2012)

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Figure captions

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Figure 1. Schematic representation of the adsorption processes

Figure 2. AFM images of ssDNA-SWNT and dsDNA-SWNT hybrids with and without SSB

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molecules

(a) ssDNA-SWNT without SSB. (b) Mixture of ssDNA-SWNT and SSB protein. (c) Height

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distribution of ssDNA-SWNT hybrids observed in (a) and (b). (d) dsDNA-SWNT without SSB. (e) Mixture of dsDNA-SWNT and SSB protein. (f) Height distribution of dsDNA-SWNT hybrids

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observed in (d) and (e). Average values of the diameters were 1.58 ± 0.37, 1.69 ± 0.58, 4.93 ± 1.68, and 1.75 ± 0.66 nm for ssDNA-SWNT without SSB, dsDNA-SWNT without SSB, ssDNA-SWNT

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with SSB, and dsDNA-SWNT with SSB, respectively. Three hundred cross-sections of 100

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molecules (3 positions for one hybrid) were measured for each sample.

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Figure 3. Images of the gels following agarose gel electrophoresis: the top and bottom images were taken under visible light and UV light after EtBr staining, respectively

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The incubation period was (a) 10 and (b) 30 min. Concentrations during incubation was as follows. ssDNA: 400 μg/ml in lanes 1–6. SWNT: 400 μg/ml in lanes 1–6. SSB proteins: 0, 25, 50, 75, 100, and 500 μg/ml in lanes 1–6, respectively. dsDNA: 400 μg/ml in lanes 7–12. SWNT: 400 μg/ml in lanes 7–12. SSB proteins: 0, 25, 50, 75, 100, and 500 μg/ml in lanes 7–12, respectively.

Figure 4. Agarose gel electrophoresis The CBB solution was added to the loading samples before the electrophoresis, and the gel was observed under visible light after the electrophoresis. The incubation period was (a) 10 and (b) 30 min. Concentrations during incubation was as follows. ssDNA: 400 μg/ml in lanes 1–6. SWNT: 400 μg/ml in lanes 1–6. SSB proteins: 0, 25, 50, 75, 100, and 500 μg/ml in lanes 1–6, respectively. dsDNA: 400 μg/ml in lanes 7–12. SWNT: 400 μg/ml in lanes 7–12. SSB proteins: 0, 25, 50, 75, 100,

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and 500 μg/ml in lanes 7–12, respectively.

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Highlights

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/SSB proteins selectively bound to ssDNA-SWNT hybrids. /SSB proteins rarely bound to dsDNA-SWNT hybrids /DNA molecules on SWNT surfaces retained their structures and properties. /Electrophoresis procedure was modified for examining SSB-DNA-SWNT hybrids.

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Graphical Abstract (for review)

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SSB-ssDNA-SWNT

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100 nm

SSB-dsDNA-SWNT

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100 nm

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Figure(s)

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ssDNA

SSB protein Hybridization with sonication

37℃ 10 or 30 min

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SWNT

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Figure 1 Nii et al.

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dsDNA

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300

(d) Lateral position [nm]

250 200 150

50 0

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0 4 8 [nm]

250 200 150 100 50 0

80 60 40 20 0

0 4 8 [nm]

dsDNA-SWNT

9.5

8.5

7.5

5.5

4.5

SSB-dsDNA-SWNT

3.5

9.5

0

Height [nm]

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Height [nm]

8.5

7.5

pt 6.5

5.5

4.5

3.5

2.5

1.5

0.5

SSB-ssDNA-SWNT

Lateral position [nm]

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Counts

ssDNA-SWNT

2.5

80 60 40 20 0

50

(f) dsDNA-SWNT hybrids

1.5

(c) ssDNA-SWNT hybrids

100

300

(e)

0 4 8 [nm]

150

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400 350 300 250 200 150 100 50 0

0 4 8 [nm]

0.5

Lateral position [nm]

(b)

200

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100

250

6.5

Lateral position [nm]

(a)

300

Figure 2 Nii et al.

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(a) ssDNA+SWNT

(b) ssDNA+SWNT

dsDNA+SWNT 1 2 3 4 5 6 7 8 9 10 11 12

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dsDNA+SWNT 1 2 3 4 5 6 7 8 9 10 11 12

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Vis

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UV

E. coli SSB concentration

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E. coli SSB concentration

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Figure 3 Nii et al.

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(b) ssDNA-SWNT

dsDNA-SWNT

7 8 9 10 11 12

1 2 3 4 5 6

7 8 9 10 11 12

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1 2 3 4 5 6

dsDNA-SWNT

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(a) ssDNA-SWNT

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Vis

E. coli SSB concentration

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E. coli SSB concentration

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Figure 4 Nii et al.

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