Flexibility and thermal dynamic stability increase of dsDNA induced by Ru(bpy)2dppz2+ based on AFM and HRM technique
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Ru(bpy)2dppz2+ has been widely used as a probe for exploring the structure of double-stranded DNA (dsDNA). The flexibility change of DNA helix is important in many of its biological functions but not well understood. Here, flexibility change of dsDNA helix caused by intercalation with Ru(bpy)2dppz2+ was investigated using the atomic force microscopy. At first, the interactions between ruthenium complex and dsDNA helix were characterized and the binding site size (p = 2.87 bp) and binding constant (Ka = 5.9 * 107 M−1) were determined by the relative extension of DNA helix using the equation of McGhee and von Hippel. By measuring intercalator-induced DNA elongation and the mean square of end-to-end distance at different molar ratios of Ru(bpy)2dppz2+ to dsDNA, the changes of persistence length under different ruthenium concentrations were determined by the worm-like chain model. We found that the persistence length of dsDNA decreased with increasing Ru(bpy)2dppz2+ concentration, demonstrating that the flexibility of dsDNA obviously enhanced due to the intercalation. Especially, the persistence length changed greatly from 54 to 34 nm on changing the molar ratio of ruthenium to dsDNA from 0 to 0.2. We speculated that the intercalation of dsDNA with Ru(bpy)2dppz2+ resulted in local deformation or bending of the DNA duplex. In addition, the thermal dynamic stability of DNA helix was measured with high resolution melting method which revealed the increase in thermal dynamic stability of DNA helix due to the ruthenium intercalation.
KeywordsDNA flexibility Thermal dynamic stability Ruthenium complex Atomic force microscopy
atomic force microscopy
high resolution melting
deoxyribose nucleic acid
binding site size
DNA base pairs
Ru(bpy)2dppz2+ to DNA base pairs
the concentration of ruthenium
the concentration of DNA base pairs
the ratio between the bound intercalator and the total concentration of DNAbps
the root mean square of end-to-end distance
the persistence length
the contour length
Many different techniques have been used to determine the interaction between ruthenium and dsDNA [7, 9, 13, 19, 20, 21]. Single molecule stretching experiments and atomic force microscopy (AFM) are complementary techniques for measuring the interaction between metal complexes and dsDNA [21, 22]. The binding mode and binding constant can be determined by the single molecule stretching measurements, but normally these are force-dependent at non-equilibrium state. AFM is a powerful technique to study the biophysical properties of single DNA molecules, including DNA flexibility, and it provides the advantage of direct observation of the DNA molecules when adsorbed onto supporting surfaces [23, 24, 25, 26, 27]. Several AFM studies have evaluated the DNA persistence length and the conformational state of the nucleic acid polymer confined to an imaging plane [25, 28, 29]. However, few studies have reported the physical property change of DNA duplex induced by the Ru(bpy)2dppz2+ intercalation which could influence the biological process. In the present study, AFM was employed to characterize the change of conformation and contour length of dsDNA, and the variation of persistence length of dsDNA induced by ruthenium intercalation was calculated with worm-like chain model in two dimensions. The flexibility of dsDNA induced by the intercalation of Ru(bpy)2dppz2+ was obtained by calculating the persistence length change. Besides that, melting temperature (Tm) measurements were performed with high resolution melting (HRM) method to understand the thermal dynamic stability of dsDNA after ruthenium intercalation. Understanding the changes in DNA helix induced by ruthenium intercalation will be very useful to design the novel and effective anti-cancer drugs.
Materials and methods
AFM sample preparation and imaging
Linearized plasmids (PBR322, 4361 base pairs) was purchased from Sangon Biotech and used without further purification. Ruthenium complex and dsDNA duplex were mixed in a buffer (10 mM Tris–HCl and 10 mM MgCl2, pH 7.0) with different molar ratios of ruthenium complex to dsDNA base pairs (0.1, 0.2, 0.33, 1, 2 and 3) whereas the final concentration of dsDNA was always maintained to be 0.5 ng/μL. 10 μL mixed solution was added onto the freshly cleaved mica and gently rinsed with 4 mL deionized water to remove extra divalent cations and gently blow-dried with nitrogen. The as-prepared sample was used for imaging at room temperature in air with Multimode IIIa AFM (Vecco Instruments, American). A series of 5 × 5 µm2 AFM images were captured under the tapping mode at 1 Hz scanning rate to avoid dragging of DNA by the tip. In our study, randomly curved and non-crossing DNA molecules were chosen and analyzed from many different images. Additionally, DNA molecule could be compressed under tapping mode AFM. In order to avoid the influence of tapping mode AFM, the measurements of relative length variance were performed, and the the length variance of different DNA molecule (pure DNA molecule is as control) were characterized under the same experimental conditions, so the tapping mode AFM had little influence on the experiments results.
Tm measurement of dsDNA using HRM method
DNA oligonucleotides were synthesized and purified by iPAGE (Invitrogen, Shanghai, China). The ratios of ruthenium complex to dsDNA were maintained consistent for AFM experiments and measured the Tm with the well mixed solution. The temperature increased gradually from 35 to 99.5 °C and fluorescence data was collected with every 0.1 °C increment. All fluorescence data were acquired by at least two parallel tests, and the measurements of melting profiles were repeated at least twice. The specific informations are shown in supplementary materials.
Results and discussion
Measurement of flexibility change of dsDNA using AFM method
Tracing and image analysis
Morphology and binding property analysis
It was feasible to derive the relative extension of DNA double stand as a function of ruthenium concentration per base pair concentration from the contour length measurement, and the results are plotted in Fig. 4. Ka of ruthenium complex and dsDNA interactions was estimated using the comparison between the experimental data and the equation converted from the theory developed by McGhee and von Hippel in the case of non-cooperative ligand binding . Figure 4 shows the comparison between our experimental data and theoretical model. The best fitting data was obtained for the p and Ka values of 2.87 bp and 5.9 * 107 M−1, respectively. These values are in good agreement with the values obtained by Williams et al. which was measured by stretching experiments with classical intercalator. In addition, as indicated by the plot of Fig. 4, the measured contour length of DNA increased with increasing Ru/DNAbps ratio. For the ratios of Ru/DNAbps is over 1, the observed contour length became saturated and the plateau indicated the 50% increase of the relative length. We speculated that ruthenium intercalation induced a change in the local structure of DNA helix resulting in a lengthening of the DNA strand. Which was consistent with previous results of classical intercalators [29, 31]. In cells, many DNA-distorting proteins used side chain intercalation to distort the DNA backbone which plays important roles for processing information in DNA and organizing chromosome DNA .
The flexibility of dsDNA analysis
In this part, we mainly presented the physical property of probed DNA molecules and how the binding of Ru(bpy)2dppz2+ affects the extension and the flexibility of DNA molecules. The flexibility of DNA can be characterized by its persistence length. Normally, there are two major methods to estimate the persistence length of adsorbed macro-molecules on the mica surface. The first method is based on the measurements of the end-to-end distance and the contour length which is very reliable when used on a large number of molecules . The second method is based on the measurements of the angle between two small segments separated by a certain distance. This method is very sensitive to the local bending of the DNA molecules and does not require measurements of hundreds of molecules. However, AFM tip can easily lead to the local bending along the scanning direction. Herein, the first method was employed to measure the persistence length of dsDNA.
Finally, by comparing the best fitted results with the equation of McGhee and Von Hippel, the value of p was found to be 2.87, which simply meant that the saturated intercalation of Ru(bpy)2dppz2+ occurred after every 3 base pairs. In other words, the persistence length would change in a small range when the intercalation reached saturation. This was in good agreement with the persistence length results in Ru/bps solution containing ratio of Ru(bpy)2dppz2+ from 1 to 3, where the ruthenium intercalation reached saturation and the persistence length decreased to approximately 20 nm.
Thermal dynamic stability of dsDNA measurement with HRM method under different ratios of Ru/DNAbps
In present study, we used AFM and HRM methods to carefully measure the change of persistence length and thermal dynamic stability of DNA helix by changing the molar ratios of ruthenium compound to DNA base pairs. The persistence length of DNA in our study decreased from 54 to 20 nm and the melting point of dsDNA increased significantly from 84.7 to 89.9 °C after Ru(bpy)2dppz2+ intercalation. Based on these two kinds of measurement results, we speculated the obvious decrease of the persistence length due to that the intercalation of ruthenium complex partially lead to detwisting along the axis of DNA helix (local deformation) and bending of DNA helix. The decrease of persistence length also suggested that the flexibility of DNA strand increased after ruthenium intercalation from the point view of the mechanical properties of materials, which agreed well with results reported by Williams et al. . In addition, the ruthenium intercalation increased the thermal dynamic stability by increasing the interaction area between two consecutive base pairs. These two kinds of results obtained with AFM and HRM can be very helpful to design the new ruthenium complex for anti-cancer drug development.
We thank Prof. Yan Jie (National University of Singapore) for his helpful discussions and Dr. Parveen Kumar for his contribution to the English editing of this article.
FCJ designed and performed the AFM experiments, contributed to data interpretation and manuscript writing. PH participated all the discussion of manuscript. KZH is responsible for the process of AFM images. JW carried out the HRM measurements. XGL analyzed the results of HRM measurements. BL revised the whole manuscript and made critical discussion of AFM experiments. All authors read and approved the final manuscript.
This study was supported by the doctoral foundation of Shandong University of Technology (No. 4041-414064).
The authors declare that they have no competing interests.
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