Single molecular study on interactions between avidin and DNA

Abstract

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.