Molecular-scale visualization and surface charge density measurement of Z-DNA in aqueous solution

The DNA in the left-handed conformation (Z-conformation) was first discovered by A. Rich, who revealed the crystalline structure of a DNA oligomer d(GC)3 by X-ray diffraction method. Later it was also found that DNA molecules change their conformations from typical right-handed form (B-DNA) to the left-handed form (Z-DNA) under specific conditions (B–Z transition). Furthermore, the detailed structures of the interface between B- and Z-DNAs, B-Z junction, was also determined with an atomic resolution. Recently it was found that some proteins have the Z-DNA binding domains, but the biological functions of Z-DNA are not well understood yet. Therefore the investigation of Z-DNA under physiological conditions is highly essential. In this study, we demonstrated the high-resolution real-space imaging of DNA molecules having the Z- and B-form conformations by frequency-modulation atomic force microscopy (FM-AFM), that has made a great progress in recent years, in an aqueous solution. The major and minor grooves of both DNA conformations were clearly visualized. Furthermore, the surface charge density was measured by three-dimensional (3D) force mapping method. We found that Z-form region was less negatively charged than the B-form region.


Results
High-resolution imaging of isolated Z-DNA. Figure 1a shows a topographic image of a plasmid DNA and Z-DNA molecules consisting of d(GC) 36 and dA 32 in 50 mM NiCl 2 . In the experiments, the B-and Z-DNAs were adsorbed onto a substrate at the same time to compare the structure of the Z-DNA to that of the B-DNA. We used the high concentration NiCl 2 solution as the rinsing and imaging solutions such that the DNA oligomers changed their conformations from the B-form to Z-form. A cross-sectional profile along the A-B polyline in Fig. 1a is shown in Fig. 1b www.nature.com/scientificreports www.nature.com/scientificreports/ that of the Z-DNA was 1.8 nm 29 . The measured heights of the molecules were lower than their diameters probably because the tip did not reach the substrate because of the adsorbates as well as the structured water molecules on the substrate. We found periodic features with a different periodicity on the B-and Z-DNA molecules. Figure 1c,d show the enlarged images of the area indicated by the white solid rectangle in Fig. 1a and a cross-sectional profile along the helix axis of the plasmid DNA in Fig. 1c. The helix pitch of the B-DNA was 3.6 nm, which is consistent with a previous study 23 . There are two kinds of grooves; i.e., wide and narrow grooves in one helix pitch. The wide grooves, referred to as the major grooves, are indicated by the red arrows, and the narrow grooves, the minor grooves, are indicated by the blue arrows. Figure 1e,f show the enlarged images of the area indicated by the white dotted rectangle in Fig. 1a and a cross-sectional profile along the helix axis of the Z-DNA in Fig. 1f. The left-handed structure was clearly resolved. The helix pitch of the Z-DNA was about 4.5 nm, which was consistent with the value measured by X-ray crystallography 27 . We also found shallow deep and shallow grooves in the Z-DNA as shown in Fig. 1f. In order to interpret the result, we simulated the AFM image of the Z-DNA using a tip model with a radius of 0.3 nm. A molecular model of the Z-DNA is shown in Fig. 1g. The simulated AFM image for this molecular model is presented below. It has been revealed by the X-ray diffraction that there is a deep groove in the Z-DNA which is an analogue of the minor groove of the B-DNA 3 , while the groove analogous of the major groove of the B-DNA is very shallow. Hereafter they are referred to as the minor and major grooves of the Z-DNA, respectively. Figure 1h shows a cross-sectional profile along the helix axis, indicated by the dotted line, of the simulated AFM image, which shows the deep (minor) and shallow (major) grooves within a helix pitch, as indicated by the blue and red arrows, respectively.
A high-resolution FM-AFM image of a DNA having the B-Z junctions with methylation (B-Z-B DNA) is shown in Fig. 2a. The helical structures of the B-form dsDNA and Z-form dsDNA were clearly visualized in both the Z-form part in the center and the B-form parts at both ends, respectively. A cross-sectional profile along the A-B polyline is shown in Fig. 2b. The red and blue arrows in Fig. 2a,b indicate the major and minor grooves of the B-DNA and Z-DNA, respectively. The helical pitch of the Z-DNA is about 4.9 nm that well corresponds to the isolated Z-DNA (Fig. 1e,f). The width of the deep groove is 3.2 nm and that of the shallow groove is 1.7 nm. In addition, the difference between the heights of the B-DNA and Z-DNA is about 0.3 nm, which is also consistent with the results shown in Fig Figure 3b shows the constant ∆f image (∆f = +110 Hz) reconstructed from the 3D frequency shift map, which corresponds to the topographic image. A cross-sectional profile along the A-B polyline in Fig. 3b is shown in Fig. 3c. The profile shows that the height in the middle region (green shaded region) of the DNA was lower than that of the outer regions of the DNA (blue shaded region), suggesting that the DNA conformations in the middle part was Z-DNA, while the outer regions were B-DNA. In spite of cytosine consisting of the Z-DNA region not being methylated, the B-Z transition was confirmed because of high ion concentration condition.

Discussion
We obtained the 3D map of the frequency shift in the volume including the interface of the B-Z-B DNA and the solution. Although the surface charges are screened by the surrounding counter ions in aqueous solutions, which form an electric double layer (EDL), the surface charge density can be measured by analyzing the EDL force between the tip and sample. We first converted the recorded frequency shift curves to the force curves by using the Sader method 30 to obtain the 3D force map, which is a collection of the site-specific force versus distance curves between the tip and sample. We used a simple equation of the EDL force between two spheres assuming www.nature.com/scientificreports www.nature.com/scientificreports/ the tip and DNA as spheres to estimate the surface charge densities of the DNA under the tip, and calculated the linear charge density of the DNA. The surface charge density of the silicon oxide tip (σ t ) in the aqueous solution used in this experiment was assumed to be −1.8 mC/m 2 31 . On the other hand, the surface charge density of the B-and Z-DNAs (σ s ) should be on the order of −100 mC/m 2 when the phosphate groups are fully dissociated 26 . Therefore we used the equation of the EDL force between the tip and sample under the condition that the surface charge density of the DNA is much higher than that of the tip (σ s ≫ σ t ), where R s and R t are the radii of the DNA molecule and the tip, respectively, λ D is the Debye length, ε is the relative dielectric constant, ε 0 is the dielectric constant of a vacuum, and z is the distance between the two spheres. We set R s as 1 nm for the data on the B-DNA and 0.9 nm for the data on the Z-DNA. The tip radius (R t ) was assumed to be 7 nm (manufacturer value). We neglected the contribution of the EDL force between the tip and the substrate to the force curves recorded on the DNA because the tip-substrate distance (DNA diameter) was higher than the Debye length (0.79 nm) in the 50 mM NiCl 2 solution. Therefore, we only considered the EDL force between the tip and the DNA. For the force curves recorded on the substrate, we used the equation of the EDL force between the spherical tip and the substrate plane, which is the reduced form of Eq. (1) under the condition R s ≫ R t . We obtained the surface charge density map by fitting Eqs (1) and (2) to force curves on the DNA molecule and the surface, respectively, as shown in Fig. 4a.
To exclude the contribution of the van der Waals force, the non-shaded region was used for the fitting (see Supplementary Fig. 2 for the original force curves). Figure 4b shows a surface potential profile along the A-B polyline in Fig. 4a. It was clearly found that the surface charge density of the B-DNA was greater than that of the Z-DNA. We averaged force curves on the B-DNA and Z-DNA, and determined the surface charge density of the B-DNA and Z-DNA as −163 mC/m 2 and −116 mC/m 2 , respectively. Note that the surface charge density of  www.nature.com/scientificreports www.nature.com/scientificreports/ the B-DNA was in quite good agreement with a theoretical value (-150 mC/m 2 ) considering that the phosphate groups are fully dissociated 26 .
We consider that the difference in the surface charge density depending on the conformation predominantly originates from the differences in the helical pitch. Since the Z-DNA molecules have slightly stretched structure compared with the B-DNA, the distance between base pairs for Z-DNA is higher than that for B-DNA. In the previous studies, the linear charge densities of B-DNA and Z-DNA were estimated by assuming the full dissociation of the phosphate groups 15,16 ; Two elementary charges per base pair length (0.34 nm and 0.37 nm) gives the linear charge densities of B-DNA and Z-DNA as −942 pC/m and −866 pC/m, respectively 15 . In this case, the linear charge density of Z-DNA is lower than that of B-DNA by about 8%. Since the linear charge density of a cylinder having a radius of R s and a surface charge density of σ s is calculated as 2πR s σ s , the linear charge densities of the Band Z-DNAs in our measurement are calculated as −1024 pC/m and −656 pC/m, respectively. Our measurement showed that the linear charge density of Z-DNA is lower than that of B-DNA by about 36%. One of the possible reasons for the discrepancy is the difference in the location of the negatively charged oxygen. The distance of the charged oxygen from the center is reported as 0.95 nm and 0.715 nm for B-and Z-DNAs, respectively 15 , and the EDL force measured at the molecular surface is smaller when the charge is located deeper in the molecule.
We demonstrated the molecular-scale visualization and the surface charge density measurement of the Z-DNA in an aqueous solution by using FM-AFM. The isolated short Z-DNA as well as the plasmid DNA were clearly visualized. The deep and shallow grooves of the Z-DNA were differentiated. We also visualized a DNA having the B-Z junctions with methylation. We again observed the characteristic left-handed helical structures of the Z-form DNA. We then performed a 3D force mapping on the DNA oligomer containing the B-form region and Z-form region. By detecting the EDL forces, the linear charge density of the Z-DNA was measured as −656 pC/m, which is 64% of the B-DNA value.
We believe that these results lead to understanding of the function of the DNA molecules, not only the Z-DNA but also the B-DNA, since the conformation and the surface charge density play an important role in the DNAprotein recognition. The conformational change of the DNA molecules induced by guanine-rich sequence is related to the biological functions of the DNA such as gene expression and regulation. Therefore, it is important to visualize the structure of non B-DNA conformations for understanding the functions of the DNA in biological systems. The results presented are the first demonstration of the capability of AFM to measure the surface charge density distribution in a single biomolecule. Therefore, AFM can be used for nanometer-scale investigations of structures and basic properties of biomolecules and their complex, such as the DNA-protein complex, in physiological solutions.

Methods
Isolated Z-DNA and plasmid DNA. Single-stranded DNA oligomers consisting of dA 32 and d(GC) 36 , were commercially synthesized by BEX. The DNA oligomers were dissolved in a TNE buffer solution (10 mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 8.0) at a concentration of 9 µM. The oligomer solution was heated to 77 °C, then cooled to 20 °C over 1 hour. After annealing, the DNA solution was diluted to a concentration of 90 nM with a TE buffer solution. The plasmid DNA (pUC18, 2686 base pairs) molecules were purchased from Takara Bio. The plasmid DNA molecules were dissolved in the TE buffer to a concentration of 5 ng/µl. Five µl droplets of the Z-DNA solution, plasmid DNA solution, and a solution containing 50 mM nickel(II) chloride hexahydrate (NiCl 2 ·6H 2 O, 99.9998% purity, Alfa Aesar) were deposited onto a fleshly cleaved muscovite mica substrate (Furuuchi Chemical). After waiting for five minutes, the substrate was rinsed by the 50 mM NiCl 2 solution, and imaged in the same solution without drying the sample. The UCSF Chimera package (Resource for Biocomputing, Visualization and Informatics, University of California, San Francisco) 32 was used to generate a graphical representation of the Z-DNA ( Fig. 1(g)).
DNA having B-Z junctions with methylation. Complementary single-stranded DNA (ssDNA) oligomers each having d(G 5me C) 12 at the center and a random sequence at both ends were commercially synthesized www.nature.com/scientificreports www.nature.com/scientificreports/ by Eurofins Genomics (see Supplementary Information). Cytosine bases in poly d(G 5me C) 12 sequences at the center were methylated because methylation of the cytosine makes the Z-form conformation stable under physiological conditions 33 . The DNA oligomers were dissolved in the TE buffer solution to a final concentration of 2 µM. The solution containing the DNA oligomers was diluted with the TNE buffer solution to a concentration of 20 nM. The DNA solution was heated to 80 °C and slowly cooled to 40 °C over 12 hours. After annealing, the DNA solution was dropped onto a fleshly cleaved mica substrate and imaged in the same manner as already mentioned.
DNA having B-Z junctions without methylation. The complementary ssDNA consisting of d(GC) 12 and the random sequence were commercially synthesized by Eurofins Genomics (see Supplementary  Information). The DNA oligomers were dissolved in the TE buffer solution to a concentration of 10 µM. The DNA solution was diluted with the TNE buffer solution to a concentration of 40 nM. The solution containing the DNA oligomers was heated to 85 °C and slowly cooled to 20 °C at the rate of −1 °C/min. After annealing, the DNA solution was dropped onto a fleshly cleaved mica substrate and imaged in the same manner as already mentioned.
FM-AFM imaging. We used lab-modified FM-AFM instruments based on a Shimadzu SPM-9600 with a home-build controller programmed in LabVIEW (National Instruments). A silicon cantilever (OMCL-AC240TN, Olympus), whose nominal second spring constant and resonance frequency in the imaging solution were 91 N/m and 150 kHz, respectively, was used. The cantilever was oscillated at its second resonance frequency and the frequency shift was detected by a digital phase-locked loop (HF2LI, Zurich Instruments). The typical oscillation amplitude was 0.5 nm peak-to-zero. WSxM (Nanotech Electronica) 34 was used to analyze the obtained data.
Simulation of topographic AFM images. GeomAFM Simulator software (version 1.1) in the SPM SimSoftware Suite 35 was used to simulate the AFM image of the Z-DNA. The AFM image was simulated by calculating the tip trajectory when the tip touched and followed the outermost atoms. The tip was modeled as a sphere with a radius of 0.3 nm and a cone with a half cone angle of 10°. The molecular structure was obtained from the Protein Data Bank (PDB ID code: 2DCG 4 ).