【作者】 王艳伟；

【导师】 满宝元； 杨光参；

【作者基本信息】 山东师范大学 ， 原子与分子物理， 2011， 博士

【摘要】 DNA的凝聚是分子生物学的核心之一，而单分子操纵技术在分子生物学的发展中提供重要的手段，本文主要是采用单分子磁镊技术（MT）与原子力显微镜技术（AFM），对DNA的凝聚过程的溶剂效应进行研究。主要研究内容包括：1.利用原子力显微镜技术（AFM），系统地研究了由乙醇与多价离子(hexammine cobalt（III） [(Co（NH₃）₆³⁺])协同作用导致的lambda-DNA凝聚现象。单独测得三价Co（NH₃）₆³⁺的临界凝聚浓度大约是10μM，乙醇的临界凝聚浓度大约是15% （v/v）。若三价离子Co（NH₃）₆³⁺的浓度大于400μM时,可以观察到DNA的解凝聚现象。在DNA溶液中同时加入乙醇（12%）与（ ）3Co（NH₃）₆³⁺（8μM），当其浓度各低于临界值时，也可观察到凝聚现象，说明乙醇与（ ）3Co（NH₃）₆³⁺对DNA的凝聚有协同作用。而且在协同作用下，可以观察到典型的圆环结构（toroids）。其中解凝聚现象可能运用电荷逆转与离子释放机制来解释。2.通过AFM与MT的技术，研究酒精导致的DNA凝聚。酒精一般被用作提纯DNA的沉淀剂，我们通过MT与AFM技术研究酒精导致的单分子DNA凝聚。首次证明酒精引起的DNA塌缩的中间亚稳球拍态的存在。对于凝聚力的测量，甚至在50%酒精浓度都不到0.2pN，这个值远远小于多价离子与表面活性剂。还证明了酒精导致的DNA形态的B-A转变，发现DNA的A形态的弹性模量比B形态的要大。与多价离子相比，酒精导致DNA凝聚的拉伸实验表现了不同的特性，其拉伸曲线的步长范围比较大，从几十纳米到几微米的范围都有，与多价离子相对统一的200nm的步矩形成对比。同时我们发现，随着酒精浓度的增加，DNA的持久长度成单调减小的趋势。在弱溶剂里，由于DNA片段之间的弱相互作用引起的DNA的凝聚形态，多是一些不规则球拍组成的松散花状的结构。通过AFM成像进一步证实这些现象与分析结果。得出结论，在酒精导致的DNA凝聚中溶剂排斥效应比电荷中和效应更占据主导地位。3.多个识别位点的内切酶与DNA相互作用的方式有很多，其中通过结合成环状的方式在许多基础生物化学的过程中起着很重要的作用。限制性内切酶BspMI必须绑定DNA上面两个有效的识别位点才具有有效的活性,这是研究这种成环相互作用的有用模型。在实验中Ca²⁺代替正常的内切酶辅助因子Mg²⁺,限制了内切酶的切割作用,导致内切酶与DNA的结合,而且这种内切酶一般是通过四聚体的形式与DNA的一个位点结合或者通过一个四聚体结合两个位点形成环状结构。我们通过AFM研究不同浓度内切酶作用下的DNA-内切酶成像。从云母片上面的成像可以清楚看见特异性结合的内切酶在DNA上面的位置,并且通过实验观察得出非特异性结合的内切酶数量占整个BspMI-DNA复合物的比例不到8%。当酶的浓度增加,在云母表面的内切酶与DNA的结合率与成环率就会增加,但当高到一定程度时,结合位点会达到饱和。在实验过程中我们还统计了BspMI-DNA复合物的平均成环大小。更多还原

【Abstract】 DNA condensation is the core of molecular biology.Single molecule manipulation is the main method of the molecule biology. In this paper, we study DNA condensation by sinle molecule magnetic tweezer（sMT） and atomic force microscopy（AFM）. The main contents are as fellows:1. We performed systematic studies of lambda-DNA condensation on mica surfaces induced by alcohol and hexammine cobalt （III） [Co（NH₃）₆³⁺] using AFM. The critical condensation concentration for Co（NH₃）₆³⁺ was found to be about 10μM; the DNA molecules extended freely on mica when the concentration was below the critical value. The morphology of condensed DNA became more compact with increasing concentration. At about 500μM Co（NH₃）₆³⁺ concentration, no condensation patterns could be observed due to charge inversion of the compact structures resulting in failure of adhesion to the positively charged surfaces. The critical concentration for alcohol was about 15% （v/v）. At this concentration, a few intramolecular loops could be observed in the AFM images. With increasing ethanol concentration the condensation pattern became more complicated ranging from flower-like to pancake-like. When the solution contained both alcohol and hexammine cobalt （III）, DNA condensation patterns could be observed even when the concentrations of the two condensation agents were lower than their critical values. We observed this phenomenon by adding mixtures of 10% alcohol and 8μM hexammine cobalt （III） to DNA solutions. The condensation patterns were more compact than those of the condensation agents separately. Typical toroids were found at an appropriate alcohol and hexammine cobalt （III） concentration. The collaborative condensation phenomenon was analyzed by electrostatic interaction and charge neutralization.2. As a widely used precipitation agent for DNA extraction, ethanol is used to induce single molecule DNA condensation. This process is studied with force-measuring MT and AFM. Our experiments provide direct evidence of the metastable intermediate racquet states in DNA collapse induced by ethanol. The measured condensing force is less than 0.2pN even at 50% ethanol concentration, which is much less than those induced by multivalent cations and cationic surfactants. We confirmed the A-B transition of DNA in ethanol and found that the tensile modulus of A-form DNA is larger than that of B-form. Single molecule pulling experiment shows very different features of neutral ethanol from those of multivalent cations. The pulling curve contains a wide range of step sizes, ranging from tens of nanometers to a few micrometers, contrasting with the relatively uniform interval （about 200 nm） in multivalent cations. Meanwhile, the persistence length of DNA decreases monotonically with the increasing ethanol concentration. The condensing morphologies by the weak attraction of DNA segments in the less polar solvent are loose and flowerlike structures composed of many annealed irregular racquets. The analysis of pulling experiments is supported by AFM direct imaging. We concluded that the dominant factor in DNA condensation induced by ethanol is solvent exclusion rather than charge neutralization correlation effect.3. Proteins interacting at multiple sites on DNA via looping play an important role on many fundamental biochemical processes. Restriction endonuclease BspMI that must bind at two recognition sites for efficient activity is a useful model system for studying such interactions. In our experiments, Ca²⁺ was substituted for the normal enzyme cofactor Mg²⁺. Under these conditions it was found that specific binding occurs but cleavage does not. Enzyme in tetramer way interact DNA in the manner of binding one site or forming loops on two sites. AFM images of DNA molecules adsorbed on mica in our experiments for different BspMI concentrations. The present of the enzyme at the specific position can be easily observed. It is worth noting that nonspecific binding to DNA represent less than 8% of the total BspMI-DNA complexes. When the enzyme concentration is increased, the high binding rate and the rate of forming loops were occurred on mica surface. The statistics on loop sizes about BspMI-DNA complexes were reported in this study.更多还原