In the preset study, we report the suppression and promotion of DNA charge inversion by mixing a quadrivalent counterion (spermine) with mono-, di- and trivalent counterions by dynamic light scattering (DLS) and single molecule electrophoresis (SME) methods. We find that the electrophoretic mobility of DNA in spermine solution decreases in the presence of monovalent sodium ions and divalent magnesium ions. It means that the charge neutralization of DNA by the quadrivalent counterion is suppressed when adding extra mono- or divalent counterions. More specifically, at a high concentration of spermine, the positive mobility can switch back to a negative value by adding mono- and divalent counterions. Thus, charge neutralization and inversion of DNA by quadrivalent counterions is suppressed in the mono- and divalent ion solution. However, the scenario changes dramatically when we add trivalent ions into the solution of DNA and spermine. In this case, the charge neutralization and inversion of DNA is promoted rather than suppressed by mixing with trivalent ions. The negative electrophoretic mobility can be promoted to a positive value, which corresponds to the charge inversion, by trivalent counterions. Thus trivalent and quadrivalent counterions work cooperatively in DNA charge neutralization and inversion. This promotion also occurs when highly positively charged chitosan is introduced into the solution. We explain the observation by the counterion complexation that is related to DNA condensation, which is supported by the images of atomic force microscopy (AFM).
Charge inversion of DNA is a counterintuitive phenomenon in which the effective charge of DNA switches its sign from negative to positive in the presence of multivalent counterions. The underlying microscopic mechanism is still controversial whether it is driven by a specific chemical affinity or electrostatic ion correlation. It is well known that DNA shows no charge inversion in normal aqueous solution of trivalent counterions though they can induce the conformational compaction of DNA. However, in the same trivalent counterion condition, we demonstrate for the first time the occurrence of DNA charge inversion by decreasing the dielectric constant of solution to make the electrophoretic mobility of DNA increase from a negative value to a positive value. In contrast, the charge inversion of DNA induced by quadrivalent counterions can be canceled out by increasing the dielectric constant of solution. The physical modulation of DNA effective charge in two ways unambiguously demonstrates that charge inversion of DNA is a predominantly electrostatic phenomenon driven by the existence of a strongly correlated liquid (SCL) of counterions at the DNA surface. This conclusion is also supported by the measurement of condensing and unraveling forces of DNA condensates by single molecular MT.
We have quantitatively investigated the precipitation of DNA in different valence cations and in various ethanol concentration solution. In free ethanol condition, monovalent ion cannot induce DNA condensation and in turn to precipitation. Divalent ion can cause DNA precipitation as the concentrations of both Mg 2+ (MgCl 2 ) and Ca 2+ (CaCl 2 ) go up to a critical value of about 50 mM. After adding ethanol, monovalent ion and divalent ion can promote DNA precipitation significantly, but it has no effect on the process by trivalent ions. In monovalent ion solution of 100 mM, the critical volume ratio of ethanol is about 52% for DNA precipitation. However, the value decreases significantly to 4% in 25 mM divalent ion solution. Based on the precipitation curves, we quantitatively calculated the binding energies between the cations and phosphate group of DNA. For monovalent ions and in aqueous solution, the Na + -DNA binding energy is about −5.52 KJ⋅Mol −1 (−2.63 k B T, the precipitation temperature is 253 Kelvin) and the K + -DNA binding energy is about −4.77 KJ⋅Mol −1 (−2.27 k B T). For divalent ions and in the same condition, the Ca 2+ -DNA binding energy is about −8.25 KJ⋅Mol −1 (−3.93 k B T) and the Mg 2+ -DNA binding energy is about −8.67 KJ⋅Mol −1 (−4.13 k B T). The scenario of DNA precipitation modulation by ethanol and zwitterionic ions is demonstrated by atomic force microscopy (AFM) imaging intuitively.
Avidin is a common basic protein, widely used for connecting DNA and modified surface in single-molecule techniques of biophysics, and it can also be used as a DNA vector in gene therapy. Avidin is highly positively charged and can condense DNA in solution. Understanding the physical mechanism of its condensing DNA is a key factor to promote avidin-DNA complex to be used for many purposes, such as a probe of biomacromlecules, signal enhancer or carrier of disease diagnosis.In the present study, we use atomic force microscope (AFM), dynamic light scattering (DLS), and single molecular magnetic tweezers (MT) to systematically investigate the interaction between DNA and avidin and the underlying mechanism of DNA condensation by avidin. The conformation of DNA-avidin complex is observed and measured by AFM and we find that the condensation includes two types: one is toroidal condensation of DNA induced by avidin, the other is the condensing structure by avidin compaction. Quantitative analysis shows that the size of avidin-DNA complex decreases monotonically with the concentration of avidin increasing. However, when the concentration of avidin reaches up to a critical value of 2 ngL-1, the size of complex begins to increase suddenly with avidin concentration increasing. The phenomenon is also confirmed by the corresponding DLS measurements. For example, when the concentration of avidin increases from 0 to 2 ngL-1, the size of condensed avidin-DNA complex reduces from 170 nm to about 125 nm. In the mean while, its electrophoretic mobility changes from -2.76 (10-4cm2V-1s-1) to -0.1 (10-4 cm2V-1s-1). The negative charge of DNA is mostly neutralized by avidin. From their force spectroscopy measured by MT, it is found that the extension of DNA varies almost linearly and a few stairlike jumps appear occasionally. For example, its characteristic trend is quite similar to the one by histones. The condensing force of DNA by avidin grows up with the concentration of avidin increasing. The statistics of force-extension curves by MT shows that the peak of unraveling steps of avidin-DNA complex is around 160 nm, which corresponds to the typical toroidal structure of DNA.In DNA condensation by avidin, electrostatic interaction plays a key role due to the neutralization of negatively charged phosphate groups of DNA by cationic avidin. From the comprehensive data by AFM, DLS and MT, we conclude that the process of DNA condensation induced by avidin consists of two mechnisms: the predominant DNA-avidin electrostatic attraction and the ancillary avidin aggregation.
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