G-四链体结构的质谱光谱性质研究

发布时间：2017-12-31 18:27

本文关键词：G-四链体结构的质谱光谱性质研究　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：G-四链体是一种特殊的核酸二级结构形态,它是由富含鸟嘌呤的DNA、RNA或者核酸类似物组成的核酸高级结构。目前,人们已经获得了大量实验数据证明潜在形成G-四链体的核酸序列存在于真核生物乃至人体中,并且在生物体中起到了非常重要的生物功能调节的作用。G-四链体的构成不同于经典双螺旋结构,在双螺旋结构中,腺嘌呤(A)与胸腺嘧啶(T)通过氢键连接形成A-T碱基互补配对,鸟嘌呤与胞嘧啶通过氢键形成G-C碱基互补配对,而在G-四链体结构单元中,4个鸟嘌呤通过Hoogsteen氢键作用形成环状平面结构称为G-四分体,再通过G-四分体的相互堆积作用进一步形成G-四链体结构。G-四链体的结构受到很多因素的影响,如序列中富G序段(G-tract)长度和位置以及其他碱基的组成和数量、与序列相互作用的离子的种类以及小分子配体(ligands)与G-四链体的非共价相互作用等等。正是由于G-四链体结构受到众多内部外部因素的控制,其最终结构呈现出极其丰度的结构多态性使得研究者难以对未知序列的结构进行预测。鉴于G-四链体的结构特殊性以及G-四链体在生物体中重要的生物学意义,研究序列变化对G-四链体结构形态的影响并进一步了解结构变化规律能够指导人们认识G-四链体在生物体内的形态以及合成相应小分子药物稳定G-四链体。目前G-四链体结构的研究热点主要集中在其生物功能表达和与G-四链结构相关的大分子功能性材料的构建,对于G-四链体结构基础性的研究相对较少。由于G-四链体在结构鉴定方面的困难,目前国际主流的研究方法依然集中在利用X射线晶体衍射法和核磁共振波谱法确认和区分核酸G-四链体结构。虽然已经有一部分结构得到了解析,但是仍急需开发更多便捷快速的结构鉴定方法。利用质谱手段结合光谱技术对G-四链体结构进行鉴别的难度较大,相关研究较少,因此在这方面如果能够寻求突破,对于G-四链体结构分析将会做出重要贡献。因此,本文作者在前人的研究基础上,主要着眼于开发G-四链体结构分析中光谱法与质谱法的结合,如何将质谱的高灵敏,高通量,快速鉴定的方法结合光谱学的特性应用到G-四链体结构生物学鉴定中来,本论文的实验内容主要有如下几个方面:1.利用G-四链体的自发荧光特性研究处于不同结构状态下的G-四链体的发射荧光性质,通过对比不同类型结构产生的发射荧光,将具体结构性质与相应光谱性质进行对应,建立结构与荧光光谱特征的直接关系。前人通过实验证明平行双分子堆积G-四链体结构在260nm波长激发条件下可以产生385nm的荧光发射峰,理论计算结果证明特征峰产生的原因在于结构中堆积界面处鸟嘌呤之间芳香环堆积(aromatic overlaps)。为此我们选择了一系列富含鸟嘌呤的特征序列来研究G-四链体的自发荧光特征。被研究序列的液相结构以及结构中的鸟嘌呤堆积形态已经得到确认,我们选取合适浓度将序列充分拟合形成结构后在260nm波长下激发并考察它们的荧光发射性质。实验结果证明平行结构G-四链体可以产生特殊的荧光发射信号尤其是当结构中存在着5'-5'堆积,而且证明堆积界面的部分五/六芳香环堆积(partial 5/6-ring overlaps)不是产生385nm荧光发射的唯一方式;同时结构中的反式-反式鸟嘌呤堆积也可以产生微弱的荧光发射信号;特殊的高级多分子锁定结构还能够产生330nm和385nm的荧光发射峰。我们建立了具体结构形态和荧光发射之间的关系,对于结构进一步区分和预测具有重要意义。2.我们利用电喷雾质谱结合荧光光谱法、圆二色谱法、紫外吸光光度法以及非变性凝胶电泳法研究了互锁式双分子结构93del和其衍生序列的结构特性。通过与堆积式结构的对比我们发现,质谱法可以将互锁式与堆积式结构进行区分。我们的研究结果表明,环长度、5'-端残基、以及富G序段的位置均对互锁结构产生重要的影响。这一研究不仅揭示了部分(3+1)型互锁式结构的形成规律也进一步阐述了特定序列的形成过程。3.我们着重考察了非对称型富含鸟嘌呤碱基的序列在形成G-四链体过程中的结构规律,其中(4n-1)、(4n-2)型G-四链体中均不同程度的出现了多分子聚集的情况,通过荧光方法我们确认了这些序列形成G-四链体混杂结构。下一步我们对核酸序列进行衍生化并系统研究了富含短GG序段的DNA序列形成G-四链体后在质谱以及光谱中的相关现象。对于以上序列的气相液相实验规律的总结,我们进一步认识了G-四链体结构中的特殊作用方式。然而,现阶段无论使用G-四链体结构测试的经典方法,还是通过我们掌握的测试手段,解析文章中的某些多分子结构都有困难,因此在今后的研究中,我们也会着重开发更多了技术手段来研究G-四链体结构信息。4.我们着重研究了G-四链体核酸序列在质谱中的结构性质。为了研究高浓度下结构的转化,首先我们重新优化了仪器条件,最终通过调节四极杆离子能量和碰撞能量控制G-四链体单分子结构、双分子结构分子离子峰的丰度。我们进一步证明了在气相体系中,双分子堆积G-四链体是稳定性很好的结构形态,对质谱中电场能量的耐受性很强,不容易失去核心铵离子。其次我们研究了高单链浓度条件下非平行序列的结构转化,这种转化程度很小,即使是高分辨质谱也必须在较高能量条件下才能观察到,转化后的结构为双分子堆积G-四链体。最后我们对比了G-四链体的气相稳定性与其液相稳定性,证明二者之间可以相互印证,也为我们解释气相中结构提供了理论依据。
[Abstract]:G- four chain is a special form of nucleic acid of two level structure, which is composed of guanine rich nucleic acid structure composed of RNA DNA, a senior or nucleic acid analogues. At present, people have got a lot of experimental data to demonstrate the potential formation of nucleic acid sequence G- four chain bodies exist in eukaryotic organisms and the human body, and in the organism plays a role of the four strand.G- very important biological function of regulation is different from the classical double helix structure, the double helix, adenine and thymine (A) A-T (T) connected to form complementary base pairing through hydrogen bonding, guanine and cytosine hydrogen bonds formed by G-C base pairing, and in G- the four chain structure unit, 4 Hoogsteen guanine through hydrogen bonding to form a ring plane structure called G- four, and then through the mutual accumulation effect of G- four split further form G- four chain structure.G- four chain The structure is influenced by many factors, such as the sequence of G rich sequence segment (G-tract) length and position as well as other base composition and number of species and ion interaction and sequence of small molecule ligands (ligands) and G- four chain of non covalent interactions and so on. It is from the four G- chain structure controlled by many internal and external factors, the final structure presents the research the structural polymorphism of extremely abundance to the structure of the unknown sequence prediction. In view of the structure of G- four chain of special G- and four chain in organism important biological significance, effect of sequence variation on structure and morphology of G- four the chain body and further understand the changes of structure can help people understand the morphology of G- four chain bodies in organisms and the corresponding synthetic small molecule drug stabilized G- four chain. The current research focus of G- four chain structure of main body To construct the expression on its biological function and molecular chain structure and G- four related functional materials, for the study of G- four based chain structure of relatively less. Due to the difficulty of the four strand G- in structure identification, the current international mainstream research method is still concentrated confirmed by X ray diffraction and nuclear magnetic resonance spectroscopy in nucleic acid and distinguish the four strand G- structure. Although there have been a part of the structure have been derived, but still need to develop more efficient methods of structure identification by mass spectrometry. Methods to identify G- four chain structure combined with spectral technology is difficult, less relevant research, so in this respect if to seek a breakthrough in the G- four chain structure analysis will make an important contribution. Therefore, the author of this paper on the basis of previous studies, mainly focus on the development of spectral analysis G- four chain structure method In combination with mass spectrometry, mass spectrometry to sensitive, high-throughput, rapid identification of the binding characteristics of Applied Spectroscopy to G- four chain structure of biological identification to the contents of this paper are as follows: 1. using G- four chain autofluorescence characteristics in fluorescence emission properties of G- four the chain of different structures under the condition of the fluorescence emission produced by comparing different types of structure, the corresponding specific structural properties and the corresponding spectral properties, a direct relationship between the structure and fluorescence spectra. Previous experiments show that the parallel double fluorescent molecular packing G- four chain structure in 260nm wavelength excitation conditions can produce 385nm the emission peak, theoretical calculation results prove that the reason is the characteristic peak structure at the interface between the accumulation of guanine (aromatic overlaps) aromatic ring stacking. We chose a Autofluorescence characteristics of guanine rich sequence series to study the G- four chain body. Study the liquid phase structure and sequence structure of the guanine accumulation form has been confirmed, we will select the appropriate concentration of sequence fully fitted to form structure at 260nm wavelength excitation and investigate their fluorescence properties. The experimental results prove the parallel structure the four strand G- body can produce special fluorescent emission signals especially when the structure exists in the accumulation of 5'-5', but also that the accumulation of part of the interface five / six aromatic ring stacking (partial 5/6-ring overlaps) are not the only way to produce 385nm fluorescence emission; and trans - structure in trans guanine can also produce weak accumulation the fluorescence emission signal; advanced multi molecular special locking structure can also produce fluorescence emission peak of 330nm and 385nm. We established the specific structure and fluorescence The relationship between the emission, to further distinguish and structure prediction has important significance of.2. we use electrospray mass spectrometry combined with fluorescence spectroscopy, circular two chromatography of interlocking double molecular structure of 93del and its derivative sequence structural characteristics of UV spectrophotometry and non denaturing gel electrophoresis. By contrast with the accumulation of the structure we found that mass spectrometry can be interlocked with the stacked structure distinction. Our results show that the ring length, 5'- terminal residues, and the position of G rich segments all have important effects on the interlocking structure. This study not only revealed the formation of type (3+1) interlocking structure also further expounds the formation process of sequence specific.3. we focus on the study of asymmetric guanine rich sequences in the formation of base structure of G- four chain process, including (4N-1), (4n-2) G- type four chain The body had different degree appeared in a number of molecular aggregation, by fluorescence method we confirmed these sequences form G- four chain of hybrid structure. The next step we studied the DNA sequences of short GG segments and rich system of nucleic acid sequences derived form G- four chain in mass spectrometry and related phenomena in the spectrum. For the above sequence of gas and liquid phase experimental summary of the law, we further understand the special role of G- four chain structure. However, the classical method whether using G- structure four chain test stage, or through the test means we know, some molecular structure analysis in the article there are difficulties, so in the future study, we will focus on the development of more technical means to study the G- four chain structure information.4. we mainly study the structure of G- four chain of nucleic acid sequences in the mass spectrometry. The study on the transformation of structure under high concentration, we re optimize the conditions of the instrument, finally by adjusting the quadrupole ion energy and collision energy control of G- four single chain molecular structure, molecular structure, molecular ion abundance double peaks. We further prove that in the gas phase system, double molecular packing G- four chain structure form a good stability, strong resistance to electric energy mass, not easy to lose the core of ammonium ion. Secondly, we study the structure transformation of non parallel sequence ssDNA concentration conditions, the conversion degree is very small, even the high resolution mass must also be in high energy conditions can be observed, the structure transformation the double molecular packing G- four chain. Finally we compared the phase stability of G- four chain gas and its liquid phase stability, that between the two can confirm each other, but also for our interpretation of the gas phase structure. The theoretical basis is provided.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q52;O657.63

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