盐浓度和温度对DNA结构和特性影响的研究
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摘要
DNA(deoxyribonucleic acid)是遗传信息的载体,遗传信息由DNA转录给mRNA(message ribonucleotide),然后以之为模板翻译成特定的蛋白质序列,通过蛋白质的加工、修饰形成一定的空间构象(conformation),以执行各种生物功能。DNA及其合成材料的优良弹性使其成为制作刚性的分子手柄、操纵其他分子的理想材料,可以作为纳米器件的基本单元而引起广泛的研究兴趣。DNA的独特结构和优良性能在生物、医学、材料等学科有着巨大的应用潜力。DNA的分子结构和特性与环境因素(例如温度、湿度、pH值、溶液的盐浓度等)密切相关, 当环境条件改变时,碱基的堆积、氢键相互作用以及磷酸基团之间的斥力都将随之改变。为了深入研究DNA的特性,本论文研究了盐浓度和温度对DNA结构和特性的影响,得到了一系列有价值的研究成果。
1) 首先简单描述了DNA的结构特点以及环境因素对其结构转变的影响,然后介绍了单分子操纵技术在DNA特性研究中的最新进展。
2) 分析了DNA分子受到外力作用时氢键的变化以及盐浓度对氢键和堆积作用能的影响,通过阳离子和DNA磷酸根之间的静电相互作用,对描述氢键的Morse势和堆积作用的范德瓦尔斯势进行了相应的修正,导出了盐浓度对氢键和堆积作用影响的表达式,给出了与盐浓度有关的DNA的弹性模型,得到了力-延伸曲线随盐浓度的变化规律:随着盐浓度的增加,延伸相变力非线性增加。同时还得到了折叠角分布随盐浓度的变化规律:对特定的相变延伸力,盐浓度较高时,分布曲线主要集中在左右,分子处于B状态;随着盐浓度的降低,B状态的几率减小,S状态的几率增大;盐浓度减小到一定程度时,分布曲线主要集中在左右,分子处于S状态;盐浓度的降低使B-S的转变更加容易。
3) 创新性地给出了与盐浓度有关的非线性哈密顿模型并研究了熔解(melting)相变特性,得到DNA的比热、熵以及变性(denaturation)温度与盐浓度的关系。通过进一步考虑主链格点振动的相互作用能,给出DNA的非线性动力学模型,讨论了盐浓度对DNA变性相变产生的影响,得到了描述界面运动的扭结孤波和相变力随盐浓度的变化规律。结果表明:相变与盐浓度、温度密切相关,溶剂离子改变了DNA的热动力学特性,变性温度随盐浓度的增加而增
加;扭结孤波的宽度随盐浓度的增加而减小;DNA变性所需要的能量随盐浓度的增加而增加,相变力随之增加,盐浓度越高越不容易变性。
4) 利用拉曼光谱测量了DNA、胶原蛋白的温度效应。对DNA的拉曼光谱测量发现,几乎所有谱线强度随温度的变化都具有相同的规律,并从中得到了位于38℃、82℃的两个峰,其中82℃的相变峰与DSC(differential scanning calorimetry)的测量结果一致,38℃的峰与DNA的功能活跃区有关,在低温区没有发现相变点。当温度变化时,碱基、磷酸根等特征振动不同程度的受到影响,谱线强度和频率随温度呈非线性变化。在所有振动模式中,碱基的特征振动受到温度的影响最大,说明碱基的堆积程度与温度的变化密切相关。除了磷酸根的谱线1101cm-1以外,其余出现频率变化的谱线均随温度的升高向低波数移动,温度变化导致多数特征谱线的移动主要集中在变性的起始点70℃左右。
胶原蛋白在不同温度的拉曼光谱表明:当温度升高时,多数谱线向低波数移动,谱线1003 cm-1的波数基本保持不变,波数为1302 cm-1的谱线明显向高波数移动。通过拉曼谱线强度的温度依赖性得到了位于0℃、42℃、68℃和90℃的4个峰,其中42℃、68℃分别与DSC和SHG(second harmonic generation)的测量结果一致;0℃的峰与冰冻密切相关,90℃的峰与胶原的二级结构被破坏有关,这两个峰在其他文献中未见报道。
5) 测量了不同激发光源下红细胞的拉曼光谱,发现红细胞的拉曼谱具有明显的共振特性。通过结肠癌、乳腺癌细胞与对应的正常细胞拉曼光谱的对比,发现癌细胞的拉曼谱线强度和频率与癌细胞的状态有着明显的关系;由于细胞癌变,DNA的两个磷酸骨架峰782cm-1和1084cm-1明显减弱,说明DNA的磷酸骨架有一定的断裂,导致癌细胞的分裂繁殖失去有效的控制。癌细胞的拉曼谱线特征为癌症诊断和治疗提供了有力的实验依据。
DNA (deoxyribonucleic acid) is the carrier of genetic information. Firstly, DNA transports genetic information into mRNA (message ribonucleotide) which is taken as a template of protein and translated into special protein sequences. By procession and modification of proteins, they come into being defined space configuration in order to perform all kinds of biological functions. The excellent elasticity of DNA and its complex make it as a kind of wonderful materials to make rigid molecule handle and manipulate other molecules. As DNA is regarded as a number of DNA-based units of nano-devise, more and more scientists pay attention to this research field. The particular structure and wonderful properties of DNA have tremendous application potential in biology, medicine, material science and so on. The structure and properties of DNA are related to environment (for example temperature, humidity, pH, salt concentration, et al.). The base-stacking and hydrogen-bond interactions change as environment condition varies. In order to further investigate the properties of DNA, we study the effects of salt and temperature on the structure and properties of DNA, and obtain some important results in this dissertation.
1) Firstly, the structure characteristics and the effects of environment condition on the structure transition of DNA are described. Then the recent progresses of single molecule manipulation techniques that study the properties of DNA are introduced.
2) We analyze the change of hydrogen-bond and the effects of salt concentration on hydrogen-bond and stacking interactions when DNA molecule is under external force. On the basis of ZZO model, we give a DNA model considering the hydrogen-bond and base-stacking interactions which are related to the salt ion concentration, and then discuss the force extension curves at various salt concentrations. With the salt concentration increasing, the stretching transition force increases nonlinearly. In addition, the folding angle distributions are obtained at different salt concentrations. When salt concentration is high, the distribution curve concentrates on the B-form configuration under given overstretching forces. With the decrease of salt concentration, the probability of S state increases rapidly, while the
probability of B state decreases. When salt concentration decreases to some extent, the probability is very small for B state marking that this transition is easier to reach at low salt concentration than that at the high.
3) The novel nonlinear dynamic model concerned with salt concentration is given and the melting transition of DNA is studied. The specific heat, entropy and melting temperature of system versus salt concentration are obtained. The nonlinear dynamic model of DNA is further studied on account of the effect of phosphate backbone. The effects of salt on denaturation transition are analyzed. By studying the nonlinear dynamic equation, we obtain the kink soliton solution of equation and discuss the influences of salt on phase transition force of DNA denaturation. The results show that melting phase transition is related to salt concentration and temperature. Solvent ion changes the structure and thermodynamic properties of DNA, and the melting temperature and phase transition force increase with the rise of the salt concentration. The width of kink soliton decreases and the energy needed for denaturation increases as salt concentration rises.
4) We measure Raman spectra of collagen and DNA at different temperatures. Raman spectra of DNA denote that all of the Raman peaks nearly have the same temperature dependence, two peaks are obtained at 38℃ and 82℃. These results are consistent with experimental data obtained by DSC (differential scanning calorimetry) at 82℃. The peak at 38℃ is concerned with the biological activity region of DNA function. No phase transition point is found at low temperature region. These spectra reveal that the vibrations of bases and phosphate groups are influenced by the change of the temperature. Base is the most sen
1) 首先简单描述了DNA的结构特点以及环境因素对其结构转变的影响,然后介绍了单分子操纵技术在DNA特性研究中的最新进展。
2) 分析了DNA分子受到外力作用时氢键的变化以及盐浓度对氢键和堆积作用能的影响,通过阳离子和DNA磷酸根之间的静电相互作用,对描述氢键的Morse势和堆积作用的范德瓦尔斯势进行了相应的修正,导出了盐浓度对氢键和堆积作用影响的表达式,给出了与盐浓度有关的DNA的弹性模型,得到了力-延伸曲线随盐浓度的变化规律:随着盐浓度的增加,延伸相变力非线性增加。同时还得到了折叠角分布随盐浓度的变化规律:对特定的相变延伸力,盐浓度较高时,分布曲线主要集中在左右,分子处于B状态;随着盐浓度的降低,B状态的几率减小,S状态的几率增大;盐浓度减小到一定程度时,分布曲线主要集中在左右,分子处于S状态;盐浓度的降低使B-S的转变更加容易。
3) 创新性地给出了与盐浓度有关的非线性哈密顿模型并研究了熔解(melting)相变特性,得到DNA的比热、熵以及变性(denaturation)温度与盐浓度的关系。通过进一步考虑主链格点振动的相互作用能,给出DNA的非线性动力学模型,讨论了盐浓度对DNA变性相变产生的影响,得到了描述界面运动的扭结孤波和相变力随盐浓度的变化规律。结果表明:相变与盐浓度、温度密切相关,溶剂离子改变了DNA的热动力学特性,变性温度随盐浓度的增加而增
加;扭结孤波的宽度随盐浓度的增加而减小;DNA变性所需要的能量随盐浓度的增加而增加,相变力随之增加,盐浓度越高越不容易变性。
4) 利用拉曼光谱测量了DNA、胶原蛋白的温度效应。对DNA的拉曼光谱测量发现,几乎所有谱线强度随温度的变化都具有相同的规律,并从中得到了位于38℃、82℃的两个峰,其中82℃的相变峰与DSC(differential scanning calorimetry)的测量结果一致,38℃的峰与DNA的功能活跃区有关,在低温区没有发现相变点。当温度变化时,碱基、磷酸根等特征振动不同程度的受到影响,谱线强度和频率随温度呈非线性变化。在所有振动模式中,碱基的特征振动受到温度的影响最大,说明碱基的堆积程度与温度的变化密切相关。除了磷酸根的谱线1101cm-1以外,其余出现频率变化的谱线均随温度的升高向低波数移动,温度变化导致多数特征谱线的移动主要集中在变性的起始点70℃左右。
胶原蛋白在不同温度的拉曼光谱表明:当温度升高时,多数谱线向低波数移动,谱线1003 cm-1的波数基本保持不变,波数为1302 cm-1的谱线明显向高波数移动。通过拉曼谱线强度的温度依赖性得到了位于0℃、42℃、68℃和90℃的4个峰,其中42℃、68℃分别与DSC和SHG(second harmonic generation)的测量结果一致;0℃的峰与冰冻密切相关,90℃的峰与胶原的二级结构被破坏有关,这两个峰在其他文献中未见报道。
5) 测量了不同激发光源下红细胞的拉曼光谱,发现红细胞的拉曼谱具有明显的共振特性。通过结肠癌、乳腺癌细胞与对应的正常细胞拉曼光谱的对比,发现癌细胞的拉曼谱线强度和频率与癌细胞的状态有着明显的关系;由于细胞癌变,DNA的两个磷酸骨架峰782cm-1和1084cm-1明显减弱,说明DNA的磷酸骨架有一定的断裂,导致癌细胞的分裂繁殖失去有效的控制。癌细胞的拉曼谱线特征为癌症诊断和治疗提供了有力的实验依据。
DNA (deoxyribonucleic acid) is the carrier of genetic information. Firstly, DNA transports genetic information into mRNA (message ribonucleotide) which is taken as a template of protein and translated into special protein sequences. By procession and modification of proteins, they come into being defined space configuration in order to perform all kinds of biological functions. The excellent elasticity of DNA and its complex make it as a kind of wonderful materials to make rigid molecule handle and manipulate other molecules. As DNA is regarded as a number of DNA-based units of nano-devise, more and more scientists pay attention to this research field. The particular structure and wonderful properties of DNA have tremendous application potential in biology, medicine, material science and so on. The structure and properties of DNA are related to environment (for example temperature, humidity, pH, salt concentration, et al.). The base-stacking and hydrogen-bond interactions change as environment condition varies. In order to further investigate the properties of DNA, we study the effects of salt and temperature on the structure and properties of DNA, and obtain some important results in this dissertation.
1) Firstly, the structure characteristics and the effects of environment condition on the structure transition of DNA are described. Then the recent progresses of single molecule manipulation techniques that study the properties of DNA are introduced.
2) We analyze the change of hydrogen-bond and the effects of salt concentration on hydrogen-bond and stacking interactions when DNA molecule is under external force. On the basis of ZZO model, we give a DNA model considering the hydrogen-bond and base-stacking interactions which are related to the salt ion concentration, and then discuss the force extension curves at various salt concentrations. With the salt concentration increasing, the stretching transition force increases nonlinearly. In addition, the folding angle distributions are obtained at different salt concentrations. When salt concentration is high, the distribution curve concentrates on the B-form configuration under given overstretching forces. With the decrease of salt concentration, the probability of S state increases rapidly, while the
probability of B state decreases. When salt concentration decreases to some extent, the probability is very small for B state marking that this transition is easier to reach at low salt concentration than that at the high.
3) The novel nonlinear dynamic model concerned with salt concentration is given and the melting transition of DNA is studied. The specific heat, entropy and melting temperature of system versus salt concentration are obtained. The nonlinear dynamic model of DNA is further studied on account of the effect of phosphate backbone. The effects of salt on denaturation transition are analyzed. By studying the nonlinear dynamic equation, we obtain the kink soliton solution of equation and discuss the influences of salt on phase transition force of DNA denaturation. The results show that melting phase transition is related to salt concentration and temperature. Solvent ion changes the structure and thermodynamic properties of DNA, and the melting temperature and phase transition force increase with the rise of the salt concentration. The width of kink soliton decreases and the energy needed for denaturation increases as salt concentration rises.
4) We measure Raman spectra of collagen and DNA at different temperatures. Raman spectra of DNA denote that all of the Raman peaks nearly have the same temperature dependence, two peaks are obtained at 38℃ and 82℃. These results are consistent with experimental data obtained by DSC (differential scanning calorimetry) at 82℃. The peak at 38℃ is concerned with the biological activity region of DNA function. No phase transition point is found at low temperature region. These spectra reveal that the vibrations of bases and phosphate groups are influenced by the change of the temperature. Base is the most sen
引文
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