抗生物素蛋白与DNA相互作用的单分子研究

Single molecular study on interactions between avidin and DNA

抗生物素蛋白(avidin)在生物单分子实验中被广泛用于DNA与修饰表面的连接,同时avidin也可作为一种DNA载体用于基因治疗中.本文利用原子力显微镜(AFM)、动态光散射(DLS)、单分子磁镊(MT)技术系统地研究了avidin与DNA之间的相互作用,以及avidin引起DNA凝聚的机理.首先通过AFM对avidin-DNA复合体形貌进行观察,发现不但有avidin导致DNA凝聚的环状形貌,同时也存在avidin自身聚集引起的DNA凝聚现象,通过定量分析,发现其凝聚尺寸越来越小,而当avidin浓度大于2 ng·μL^(-1)时,其凝聚尺寸又突然变大.DLS实验结果也显示了同样的规律,伴随着avidin浓度的升高,DNA的粒径大小从大约170 nm减小到125 nm左右,其电泳迁移率由-2.76(10^(-4)cm^2·V^(-1)·s^(-1))变化到-0.1(10^(-4)cm^2·V^(-1)·^(-1)).此外,通过MT技术的力谱曲线变化,发现avidin导致的DNA凝聚与其他多价离子相比,长度的变化曲线几乎呈线性变化,偶尔存在少而小的阶跃,这种变化趋势与组蛋白的变化曲线更相似.因此可以判断,avidin导致DNA凝聚是由avidin与DNA的静电吸引和avidin自身聚集两种相互作用引起的.

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 ng·μL-^（-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 ng·μL-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^（-4）cm2·V^（-1）·s^（-1）） to-0.1（10^（-4）cm^2·V^（-1）·s^（-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.