In this study, we investigated the behavior of DNA on supported lipid bilayers with different surface charge density and under different ionic strength. When the negatively charged DNA is adsorbed on the positively charged lipid bilayers deposited on the glass substrate, the fluidity of the lipids allows DNA to diffuse in a two-dimensional manner. It has been observed that the diffusion behavior of DNA on a two-dimensional plane is consistent with the Rouse model (D~1/N), and its radius of gyration is also consistent with the prediction given by the self avoiding random walk (~N^2v). However, there still lacks indepth study on the diffusion mechanism of DNA on the positively charged lipid membrane.
We used fluorescence microscope to track the diffusion of DNA and the radius of gyration on a two-dimensional plane. By image analysis of the position of the centroid of DNA over time, we found that DNA conducts simple diffusion over a long delay time on lipid bilayers, but sub-diffusion over a short delay time. From experimental images, we found that a DNA often has few fragments confined at fixed points, called the sticky points. DNA fragment is not completely fixed on a sticky point, but can slide through and has a certain chance to break free. These sticky points cause sub-diffusion of DNA over a short delay time, and the increase in electrostatic attraction amplifies the impact of these sticky points. At the same time, we found that the diffusion coefficient of DNA is highly correlated with the concentration of positively charged lipids. In addition, under the conditions of high positive charge lipid concentration and low ionic strength, DNA may not be able to expand after adsorption on the bilayers. We believe that these sticky points appear because the surface of the substrate is uneven at the molecular level, which causes unevenness on the surface of the lipid bilayer. For DNA, the concaved places on lipid bilayers can be regarded as electrostatic potential wells. The origin of these energy wells may be due to the following two reasons: (1) DNA located at concaved places can have a stronger electrostatic interaction with positively charged lipids due to the shape of the surface. (2) The size of hydrophilic end of positively charged lipid (DOTAP+) is smaller than that of neutral lipid (DOPC). Therefore, positively charged lipids are prone to gather in the concaved places to form potential energy wells.
In order to verify this potential well hypothesis, we experimented with another positively charged lipid with a larger hydrophilic end (EPC+). The larger hydrophilic end makes EPC+ less likely to aggregate in the concaved places, reducing the depth of energy wells. Therefore, the effect of sticky points to DNA is expected to be weakened. As a result, the diffusion coefficient of DNA is expected to increase and the phenomenon that DNA cannot be unfolded is expected to happen only at higher EPC+ concentration. In omparison with the experimental results, the diffusion coefficient of DNA on the EPC+/DOPC lipid bilayer is indeed higher than that on the DOTAP+/DOPC lipid bilayer, and the phenomenon that DNA cannot be unfolded occurs at a higher concentration. The agreement between our expectation and experiments supports the potential well hypothesis.
