实验与理论研究G-四链体中鸟嘌呤自由基阳离子脱质子反应

G-四链体传输空穴的特殊性质使其有望应用于发展分子电子器件.由于鸟嘌呤自由基阳离子（G^·+）脱质子反应会中断空穴传递,影响传递效率,我们对G-四链体AG₃（T₂AG₃）₃中G^·+脱质子过程展开了理论与实验的研究.根据瞬态紫外可见吸收光谱,确定了脱质子产物是G（N（2）-H）^·;通过测量不同温度下G^·+脱N（2）-H质子的速率常数,得到脱质子活化能为20.0±1.0 kJ/mol.进而,采用显性水和连续溶剂化模型相结合的方法模拟G-四链体中G^·+脱质子环境,在M062X/6-31G（d）水平上得到了脱质子势垒（26.4 kJ/mol）.结合实验值,理论计算的势能面描述了G-四链体中G^·+脱N2-H的过程.这些结果为G-四链体在电子器件方面的应用提供了重要依据和指导.     

色胺修饰竹红菌素及其稀土离子配位聚合物与DNA相互作用研究

利用紫外-可见吸收光谱、荧光光谱、圆二色谱(CD)等方法研究了色胺修饰竹红菌素(DTrpHA)及其稀土离子配位聚合物(Y³⁺-DTrpHA, La³⁺-DTrpHA)与小牛胸腺DNA (CT DNA)和G-四链体22AG的相互作用.结果表明, DTrpHA及其配位聚合物中的色胺基团和竹红菌素基团均参与和双链CT DNA的作用,作用方式主要为沟槽作用.与G-四链体DNA作用后, DTrpHA及其配位聚合物中的色胺基团均具有较大的减色效应(> 45%)和峰位红移(≥ 4 nm),说明色胺基团与G-四链体采用外部堆积作用方式结合;而竹红菌素基团的减色效应相对较小且无明显峰位变化,表明竹红菌素基团采用非特异性作用方式与G-四链体的环区碱基或糖-磷酸骨架结合. G-四链体22AG的构象主要为分子内反平行结构,加入DTrpHA及其配位聚合物对G-四链体22AG的构象影响较小. Y³⁺-DTrpHA比DTrpHA和La³⁺-DTrpHA与G-四链体具有更强的相互作用. Y³⁺-DTrpHA使得CT DNA的熔解温度(T_m)上升了仅1.9 ℃,而使G-四链体的熔解温度上升了13.1 ℃.荧光嵌插剂置换实验 (FID)结果表明, Y³⁺-DTrpHA对G-四链体具有良好亲和性,具有较小的^G4DC₅₀值(使噻唑橙/G-四链体体系荧光下降50%所需配体或配合物的浓度)和较高的G-四链体选择性.     

基于DNA电化学传导的可重置键盘锁

选择巯基化并含有PW17碱基序列的DNA在金电极上自组装,在10 μmol/L Pb²⁺存在下,Pb²⁺诱导自组装的DNA形成Pb²⁺稳定的G-四联体.通过微分脉冲伏安法发现Pb²⁺稳定的G四联体在-0.365 V vs.Ag/AgCl出现一Pb²⁺的还原峰.依据Pb²⁺与EDTA的强配位作用,EDTA可以与G-四联体中的Pb²⁺作用,并伴随G-四联体的构型转变为自由态的DNA.以组装在电极上的DNA为工作单元,Pb²⁺和EDTA作为输入信号,-0.365 V处的还原峰为输出信号,根据Pb²⁺和EDTA加入顺序的不同,键盘锁处于“开”或“关”的状态.在Pb²⁺与EDTA的交替作用下,G-四联体和自由态DNA可以互相转化,同时电化学的输出信号在循环5次后基本保持不变,键盘锁呈现出良好的可重置性.     

吲哚并喹啉衍生物与G-四链体相互作用的分子模拟研究

G-四链体是富含鸟嘌呤碱基的DNA序列通过氢键相互作用形成的四链螺旋结构. 通过小分子化合物诱导与稳定端粒G-四链体从而抑制端粒酶活性是一种新的抗癌策略. 为了研究一系列吲哚并喹啉衍生物与端粒G-四链体的相互作用, 探究其相互作用模式, 从而为实现基于G-四链体结构的药物合理设计提供依据, 使用分子对接的方法构建了吲哚并喹啉衍生物与G-四链体复合物结构, 在此基础上进行分子动力学模拟, 并使用线性相互作用能(LIE)方法计算了化合物与G-四链体的结合自由能. 结果表明: 化合物与G-四链体的主要相互作用方式由氢键、静电与π-π堆积作用构成, 侧链末端基团类型和侧链的长短是影响相互作用强弱的重要因素. 通过LIE方法计算的结合自由能与实验结果基本吻合, 相关度达到r²＝0.79. 并且, 基于预测的结合模式, 总结了拥有更高活性的新型吲哚并喹啉衍生物应具有的几个结构特征.     

血红素/G-四链体DNA酶的活性增强研究及其在生物传感器中的应用进展

血红素/G-四链体DNA酶是一类具有类过氧化物酶活性的DNA分子,因其具有出色的活性、易修饰性和可编程性,被广泛应用于生物传感器等领域。 本文先是简要介绍了G-四链体的结构,再主要综述了增强血红素/G-四链体DNA酶活性的策略及基于血红素/G-四链体DNA酶的生物传感器在生物标志物、微生物与生物毒素以及金属离子检测中的应用,并展望了血红素/G-四链体DNA酶的未来发展趋势。     

连接环、末端碱基及一价阳离子对G-四链体结构的影响

研究了G-四链体中的连接环(Loop)、末端碱基和一价阳离子对其结构的影响,发现在K+溶液中Loop短的序列易形成平行结构,无末端碱基时容易形成多聚体,而反平行或混合平行/反平行的G-四链体则难以形成多聚体;一价阳离子K+,NH+4和Na+促进形成平行结构及多聚体的能力依次减弱.在平行G-四链体的3’或5’端增加非G碱基,或改变阳离子使其形成非平行结构,均可抑制多聚体的形成.Loop长度影响G-四链体的热稳定性,Loop短的序列可形成很稳定的分子内结构;无末端碱基的G-四链体多聚体的稳定性低于单个G-四链体,且多聚体随着温度升高而变小.结果表明,在K+溶液中,无末端碱基的平行G-四链体序列首先形成分子内结构,然后通过π-π堆积形成多聚体;末端碱基及反平行或混合平行/反平行G-四链体中的Loop可阻碍末端堆积作用,抑制多聚体的形成.本研究为G-四链体的结构与功能研究提供了有用信息.     

Loop长度对G-四链体DNA识别和结合肿瘤细胞的能力的影响

G-四链体是由富含鸟嘌呤(G)的核酸通过π-π堆积形成的核酸二级结构.前期研究发现,G-四链体DNA对肿瘤细胞具有普遍识别和结合能力,且具有如抗肿瘤增殖等生物学活性,但G-四链体DNA的结构对其识别和结合肿瘤细胞的能力的影响还未见报道.本文采用圆二色光谱和凝胶电泳对不同连接环(loop)长度G-四链体DNA的结构和稳定...     

金属离子诱导的G-四链体凝胶体系的基本性质研究

<正>核酸是生命体内最重要的生物分子之一，具有生物相容性和低毒性等特点，常被应用在生物医药等领域¹。鸟嘌呤(G)是DNA分子中的四种碱基之一，在人体端粒末端的DNA单链上，富含丰富的鸟嘌呤，使其能够形成G-四链体结构，该结构的稳定与端粒酶的活性有关，具有非常重要的生物学意义²。此外，小分子鸟嘌呤也可以通过超分子自组装形成G-四链体结构。该结构具有结构有序和性质可调控的特点，可作为模板用于功能材料的制备，应用于组织工程等领域³。     

新型氮杂18-冠-6衍生物的合成及其与阳离子的配位性能

通过1,10-二氧-4,7,13,16-四氮杂18-冠-6 (1)与2-羟基-5-硝基苄基溴反应合成了一个四硝基酚取代的氮杂18-冠-6衍生物(2). 采用UV-Vis光谱法研究了混合溶剂中冠醚2对H^＋, Mg^2＋, Ca^2＋, Sr^2＋, Ba^2＋和Pb^2＋的光谱响应及配位性能. 结果表明, 随溶液pH值的增大, 冠醚2的最大吸收峰由319 nm逐渐红移到426 nm, 并伴随着强度的增大; 其与几种阳离子形成配合物的稳定常数随阳离子半径的增加而增大, 从而展示了对Ba^2＋和Pb^2＋高的配位能力.     

利用对G-四链体环部的构型调节进行传感器的设计

对鸟嘌呤碱基G重复序列之间连接环结构对G-四链体形成的影响进行了研究。发现在连接环较长,DNA链不易形成G-四链体的情况下,可以通过将环序列设计成双链结构的方式促进G-四链体的重新形成。这就为传感器的设计提供了一个新途径,即可以利用目标分子对环部双链的调节作用控制G-四链体DNA酶的活性。为证明这一点,在双链区域引入T-T碱基错配,破坏双链结构使DNA链不能形成G-四链体。Hg2+对T-T错配的稳定作用可以促进双链结构的形成,DNA链重新折叠成G-四链体,得到的G-四链体与氯化血红素(Hemin)结合后形成具有过氧化物酶活性的G-四链体DNA酶,据此构建了Hg2+传感器。利用此传感器可在10~700 nmol/L范围内实现Hg2+的定量检测,检出限为8.7 nmol/L。在此基础上,利用半胱氨酸可以将Hg2+从T-Hg2+-T碱基对上竞争下来的能力,设计了一种半胱氨酸的检测方法。此方法可以在20~600 nmol/L范围内实现半胱氨酸的定量检测,检出限为14 nmol/L。     

Electrospray ionization mass spectrometry probing of formation and recognition of the G‐quadruplex in the proximal promoter of the human vascular endothelial growth factor gene

The formation of the G‐quadruplex of the vascular endothelial growth factor (VEGF) gene was probed by electrospray ionization mass spectrometry (ESI‐MS). It found that cations (K⁺ and NH), CH₃OH and pH influence significantly the formation of the G‐quadruplex structure. Additionally, a perylene derivative (P3) and polydatin (P4) have shown to be potential G‐quadruplex binding agents with structurally specific recognition. Copyright © 2010 John Wiley & Sons, Ltd.     

Structural dynamics and cation interactions of DNA quadruplex molecules containing mixed guanine/cytosine quartets revealed by large-scale MD simulations.

Large-scale molecular dynamics (MD) simulations have been utilized to study G-DNA quadruplex molecules containing mixed GCGC and all-guanine GGGG quartet layers. Incorporation of mixed GCGC quartets into G-DNA stems substantially enhances their sequence variability. The mixed quadruplexes form rigid assemblies that require integral monovalent cations for their stabilization. The interaction of cations with the all-guanine quartets is the leading contribution for the stability of the four-stranded assemblies, while the mixed quartets are rather tolerated within the structure. The simulations predict that two cations are preferred to stabilize a four-layer quadruplex stem composed of two GCGC and two all-guanine quartets. The distribution of cations in the structure is influenced by the position of the GCGC quartets within the quadruplex, the presence and arrangement of thymidine loops connecting the guanine/cytosine stretches forming the stems, and the cation type present (Na(+) or K(+)). The simulations identify multiple nanosecond-scale stable arrangements of the thymidine loops present in the molecules investigated. In these thymidine loops, several structured pockets are identified capable of temporarily coordinating cations. However, no stable association of cations to a loop has been observed. The simulations reveal several paths through the thymidine loop regions that can be followed by the cations when exchanging between the central ion channel in the quadruplex stem and the surrounding solvent. We have carried out 20 independent simulations while the length of simulations reaches a total of 90 ns, rendering this study one of the most extensive MD investigations carried out on nucleic acids so far. The trajectories provide a largely converged characterization of the structural dynamics of these four-stranded G-DNA molecules.     

Triplex and quadruplex DNA structures studied by electrospray mass spectrometry

DNA triplex and quadruplex structures have been successfully detected by electrospray ionization mass spectrometry (ESI-MS). Circular dichroism and UV-melting experiments show that these structures are stable in 150 mM ammonium acetate at pH 7 for the quadruplexes and pH 5.5 for the triplexes. The studied quadruplexes were the tetramer [d(TGGGGT)](4), the dimer [d(GGGGTTTTGGGG)](2), and the intramolecular folded strand dGGG(TTAGGG)(3), which is an analog of the human telomeric sequence. The absence of sodium contamination allowed demonstration of the specific inclusion of n - 1 ammonium cations in the quadruplex structures, where n is the number of consecutive G-tetrads. We also detected the complexes between the quadruplexes and the quadruplex-specific drug mesoporphyrin IX. MS/MS spectra of [d(TGGGGT)](4) and the complex with the drug are also reported. As the drug does not displace the ammonium cations, one can conclude that the drug binds at the exterior of the tetrads, and not between them. For the triplex structure the ESI-MS spectra show the detection of the specific triplex, at m/z values typically higher than those typically observed for duplex species. Upon MS/MS the antigene strand, which is bound into the major groove of the duplex, separates from the triplex. This is the same dissociation pathway as in solution. To our knowledge this is the first report of a triplex DNA structure by electrospray mass spectrometry.     

Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G‐quadruplex aptamer and thrombin

The dynamic binding status between the thrombin and its G‐quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence‐coupled capillary electrophoresis method. A 29‐nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G‐quadruplex from unstructured 29‐nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6‐carboxyfluorescein labeled G‐quadruplex aptamer was measured. Third, the stability of G‐quadruplex aptamer–thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29‐nucleic acid thrombin binding aptamer could compete with G‐quadruplex aptamer and thus disassociated the G‐quadruplex structure into an unstructured aptamer. These data suggest that our in‐house established fluorescence‐coupled capillary electrophoresis assay could be applied to binding studies of the G‐quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available.     

Quadruplex structure of polyriboinosinic acid: dependence on alkali metal ion concentration, pH and temperature

The vibrational infrared (IR) absorption and vibrational circular dichroism (VCD) spectral changes of polyinosinic acid (polyI) as a function of alkali metal ion concentration, temperature and pH have been investigated to establish how changes in spectral features relate to the structural modifications of polyI. A single positive VCD couplet associated with the carbonyl absorption band is considered to be the signature of quadruplex structure for polyI. The disruption of the quadruplex structure with temperature increase or pH increase at low alkali metal ion concentration is evidenced by the disappearance of this positive VCD couplet. The absence of any VCD signal upon quadruplex disruption indicates that the newly formed structure lacks helical chirality and is likely to be disordered. In the presence of 1 M NaCl or 0.1 M NaCl, the heat-induced quadruplex disruption is completely reversible. A mildly alkaline environment, in the presence of 0.1 M NaCl, is not sufficient to support the quadruplex structure of polyI. Trehalose-assisted polyI film at room temperature exhibits the same quadruplex spectral signature as that seen for solution at room temperature, but the quadruplex spectral signature in the film state remains at higher temperature, unlike in solution. This indicates that the quadruplex structure of polyI in the film state resists heat-induced disruptions.     

G-quadruplexes can maintain their structure in the gas phase

Several very extended (0.5-1 micros) molecular dynamics (MD) simulations of parallel and antiparallel G-quadruplex DNA strongly suggest that in the presence of suitable cations the quadruplex not only remains stable in the gas phase, but also displays a structure that closely resembles that found in extended (25-ns long) trajectories in aqueous solution. In the absence of the crucial cations, the trajectories become unstable and in general the quadruplex structure is lost. To our knowledge, this is the first physiologically relevant structure of DNA for which very large MD simulations suggest that the structure in water and in the gas phase are indistinguishable.     

Stability and cations coordination of DNA and RNA 14-mer G-quadruplexes: a multiscale computational approach

Molecular dynamics simulations have been used to study the differences between two DNA and RNA 14-mer quadruplexes of analogous sequences. Their structures present a completely different fold: DNA forms a bimolecular quadruplex containing antiparallel strands and diagonal loops; RNA forms an intrastrand parallel quadruplex containing a G-tetrad and an hexad, which dimerizes by hexad stacking. We used a multiscale computational approach combining classical Molecular dynamics simulations and density functional theory calculations to elucidate the difference in stability of the 2-folds and their ability in coordinating cations. The presence of 2'-OH groups in the RNA promotes the formation of a large number of intramolecular hydrogen bonds that account for the difference in fold and stability of the two 14-mers. We observe that the adenines in the RNA quadruplex play a key role in conserving the geometry of the hexad. We predict the cation coordination mode of the two quadruplexes, not yet observed experimentally, and we offer a rationale for the corresponding binding energies involved.     

A New Pathway of DNA G‐Quadruplex Formation

A new folding intermediate of Oxytricha nova telomeric Oxy‐1.5 G‐quadruplex was characterized in aqueous solution using NMR spectroscopy, native gel electrophoresis, thermal differential spectra (TDS), CD spectroscopy, and differential scanning calorimetry (DSC). NMR experiments have revealed that this intermediate (i‐Oxy‐1.5) exists in two symmetric bimolecular forms in which all guanine bases are involved in GG N1‐carbonyl symmetric base pairs. Kinetic analysis of K⁺‐induced structural transitions shows that folding of Oxy‐1.5 G‐quadruplex from i‐Oxy‐1.5 is much faster and proceeds through less intermediates than folding from single strands. Therefore, a new folding pathway of Oxy‐1.5 G‐quadruplex is proposed. This study provides evidence that G‐rich DNA sequences can self‐assemble into specific pre‐organized DNA structures that are predisposed to fold into G‐quadruplex when interacting with cations such as potassium ions.     

Promoting the formation and stabilization of G-quadruplex by dinuclear RuII complex Ru2(obip)L4

The remarkable ability of a new dinuclear Ru (II) complex Ru 2(obip)L 4 [obip = 2-(2-pyridyl)imidazo[4,5- f][1,10]-phenanthroline; L = 2,2'-bipyridine] to promote the formation and stabilization of the human telomeric repeat AG3(T2AG3)3 quadruplex was reported. The experimental results indicated that Ru 2(obip)L 4 could induce the formation of an antiparallel G-quadruplex structure in the absence of metal cations. It could induce positive T m shifts of +9.4 and +5.8 degrees C in Na (+) and K (+) buffers, respectively, in which an increase in the melting temperature of the quadruplex indicated a stabilizing effect. Binding stoichiometry with the quadruplex was investigated through a luminescence-based Job plot. The major inflection point for Ru 2(obip)L 4 at x = 0.48 was observed. The data were consistent with a 1:1 [quadruplex]/[complex] binding mode, which was suggestive of a specific Ru 2(obip)L 4-quadruplex interaction with a single guanine tetrad.