硫黄素T与侧翼连接双链DNA的G-四链体高特异性作用

富G碱基的DNA序列在离子诱导下可形成G-四链体(G4),基于这一构型转化设计了大量的传感检测平台。其中的荧光检测平台是基于G4与荧光小分子的相互作用。但是,G4与荧光小分子的有效结合依赖于G4构型和体系中存在的离子种类和离子浓度,尤其是高Na⁺浓度(140 mmol·L^-1)。那么如何实现G4与荧光小分子普适性地有效结合,并不依赖于体系中的Na⁺和Na⁺浓度,是一个难题。在本研究中,以最简单的富G DNA序列凝血酶适体链TBA (thrombin binding aptamer)为例,在3’端和5’端分别增加10个碱基(TBA-10 bp),K⁺诱导TBA-10 bp形成K⁺稳定TBA (K⁺-TBA,G4)并衔接含有10个互补碱基对的双链DNA (K⁺-TBA-10 bp)。相较于K⁺-TBA,硫磺素T与K⁺-TBA-10 bp结合后的荧光强度增加了100倍,相互作用强度增加了1000倍,而且与体系中的Na⁺ (5-140 mmol·L^-1)无关。结合荧光光谱,紫外吸收光谱和圆二色光谱发现硫磺素T特异性的嵌合于K⁺-TBA和双链DNA衔接处的空腔内。有趣的是,这一结合模式不受G4构型的影响。该研究结果为研究G4与荧光小分子的有效结合提供了新视角,也为拓展G4在生物功能和生化检测领域的应用提供了实验依据。     

DNA/银纳米簇荧光探针在检测Pb2+中的应用

基于DNA/银纳米簇的荧光特性报道了一种简单、灵敏、高选择性的荧光方法检测Pb²⁺.以茎部为富G结构,环状部分为聚C结构的发夹型DNA为模板合成具有稳定荧光的银纳米簇.当加入Pb²⁺后,发夹型DNA在Pb²⁺诱导下形成G-四链体结构,破坏了发夹型DNA的构型,极大地影响了合成银纳米簇的模板结构,导致银纳米簇的荧光强度降低.Pb²⁺存在和不存在时所产生荧光强度的差异与发夹型DNA的碱基序列和茎部配对碱基数有关.依据这一现象,在优化DNA碱基序列和茎部配对碱基数的基础上,可实现100 μmol/L至100 nmol/L范围内对Pb²⁺的定量检测,检出限为10 nmol/L.该方法对Pb²⁺的检测具有较好的选择性,并可应用于实际水样中Pb²⁺的检测.检测结果与原子吸收光谱进行比对,显示出较好的一致性.     

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.     

基于硫代黄素T诱导G-四联体构成的生物传感器检测铅离子

硫代黄素T(ThT)荧光分子在自由状态下荧光强度很弱,通过在Tris-HCl缓冲液中加入Pb²⁺的适配体即富含G的DNA序列,可与ThT荧光分子形成G-四联体结构,使荧光信号迅速增强;向溶液中加入Pb²⁺,Pb²⁺与其适配体有很好的结合特异性,可生成更牢固的G-四联体结构,使ThT分子被释放出来,导致溶液的荧光强度降低,基于此可检测溶液中的Pb²⁺离子.实验中优化了缓冲溶液组成、ThT荧光分子浓度、Pb²⁺适配体浓度及反应时间等条件.结果表明,在10 mmol/L Tris-HCl(pH=8. 3,含2 mmol/L MgCl₂)缓冲溶液中,ThT荧光分子和Pb²⁺适配体的浓度分别为10μmol/L和200 nmol/L,反应10 min时,随着溶液中Pb2+浓度的增加,荧光强度减弱.Pb²⁺浓度在20～1000 nmol/L范围内时,荧光强度与Pb²⁺的浓度呈现良好的线性关系(R^...     

溶液pH和阳离子对端粒G-四链体DNA形成和结构的影响

采用电喷雾质谱和圆二色谱研究了溶液pH和阳离子对人类端粒G-四链体DNA的影响. ESI-MS和CD谱图表明, pH可以引起G-四链体DNA的构象转变和离解, 而K^＋, NH₄^＋,阳离子对G-四链体DNA的形成有着重要的促进作用.     

杂元素冠醚研究(Ⅻ)──氧、硫、硒和碲杂冠醚的液膜迁移性能比较

报道了碲杂冠醚(TeB15C5)对Na⁺、K⁺、Ag⁺和Pb²⁺的液膜迁移能力,并与相应的全氧冠醚(B15C5)、硫杂冠醚(SB15C5)和硒杂冠醚(SeB15C5)作了比较;同时以SeB15C5对K⁺的迁移为例考察了冠醚浓度和盐浓度对迁移速率的影响,结果表明,TeB15C5对Na⁺、K⁺和Pb²⁺的迁移能力比SB15C5和SeB15C5强,但对Ag⁺的迁移速率比后两者慢;SeB15C5对Ag⁺的迁移速度比SB15C5快,两者对Ag⁺的选择性都相当高,可以用来分离和回收银等贵重金属。     

聚甲基丙烯酸单层保护金纳米粒子选择性检测Pb2+

以聚甲基丙烯酸单层保护的金纳米粒子(GNPs)作为传感器, 实现了水溶液中Pb²⁺的选择性循环检测.先采用柠檬酸钠还原法获得尺寸均匀的GNPs, 再通过具有硫醇端基的聚甲基丙烯酸与金的强耦合作用, 获得了聚甲基丙烯酸单层保护的金纳米粒子(PMAA-@-GNPs).动态光散射、紫外-可见吸收光谱及透射电子显微镜表征证实了其单层结构.在Pb²⁺的诱导下, PMAA-@-GNPs溶液颜色从酒红色变为紫色并可肉眼识别.透射电子显微镜结果证实, 这种变化是由于Pb²⁺交联羧基使聚合物发生收缩, 并诱导GNPs的聚集所致.对比Pb²⁺与Hg²⁺, Mg²⁺, Cu²⁺, Zn²⁺, Na⁺, Ni²⁺, Fe³⁺, Cd²⁺, K⁺和Fe²⁺溶液颜色的变化, 证实此体系具有一定的选择性.用EDTA可夺取交联的Pb²⁺, 使PMAA-@-GNPs 的吸收峰恢复并可用于循环检测Pb²⁺.     

双金属协同调控G-三链体/血红素比色传感探针的构筑及应用

该文首次将一条富含G碱基的DNA序列(5’-CTGGGAGGGAGGGA-3’)与血红素结合,形成的G-三链体/血红素复合物具有类过氧化氢酶活性,能够催化H₂O₂的氧化反应,使反应底物2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)由无色变为绿色。通过对金属离子的筛选,发现K⁺和Sr²⁺同时存在能稳定G-三链体结构,基于此构筑了双金属协同调控的G-三链体/血红素比色传感探针,并将其用于H₂O₂的定量分析。在最佳实验条件下,溶液的吸光度与H₂O₂浓度在0.5～4.5 mmol/L范围内呈现良好的线性关系,检出限为0.07 mmol/L。将该方法用于牛奶中H₂O₂的检测,加标回收率为92.5%～103%,相对标准偏差为1.2%～5.4%。该探针具有碱基序列短、成本低廉、适用性强等诸多优势,为G-三链体功能化核酸探针的研究提供了新的思路。     

凝胶原位探测c-Myc G-四链体的结构多态性

G-四链体(G4)在某些条件下可形成聚集结构,丰富了其结构多态性,但影响了其结构与功能研究。本研究建立了对平行G4 c-Myc及衍生序列形成的分子内结构和分子间聚集体同时分离与原位探测的方法,并讨论了末端碱基对c-Myc形成聚集体的影响。首先通过荧光光谱研究了硫磺素T(Thioflavin T,ThT)与G4的相互作用,发现ThT对含不同A/T末端的c-Myc响应不同;比较典型分子间平行以及反平行单体G4,发现ThT结合分子间平行G4能使其荧光强烈激活。用ThT对凝胶中的G4条带特异性染色,再用Stains-all对所有DNA条带染色,通过ImageJ获得DNA条带经双重染色的光密度比值,发现分子间平行单体G4或分子间G4聚集体的光密度比值大于1,分子内平行G4的光密度比值约为0.5,非G4结构的光密度比值接近0;发现末端碱基影响c-Myc聚集体的形成,在K⁺溶液中增加末端碱基利于形成G4聚集体,其中T作用更强;在Na⁺溶液中只有末端含两个或以上的A利于其形成G4聚集体。本研究利用特异性与非特异性的染料组合在胶上原位探测G4构型多态性的策略,...     

金属离子在冠醚化合物为载体的液膜体系中传输的研究

液膜分离是近年才发展起来的一种新分离技术^[1]。早在1968年Tosteson^[2]就已观察到冠醚化合物在人造膜中对K⁺、Na⁺离子的选择性与天然大环化合物在细胞膜中的作用具有共同之处。Cussler^[3]曾测定了DB-18-C-6为载体的液膜中一些金属离子的传输通量。Lamb^[4]比较了以DB-18-C-6、DT-18-C-6及DKP-18-C-6为载体的氯仿液膜中Pb²⁺/Na⁺、Pb²⁺/Ca²⁺、Pb²⁺/Fe³⁺、Pb²⁺/Zn²⁺的选择比。     

Cation-mediated energy transfer in G-quadruplexes revealed by an internal fluorescent probe

A single pyridine unit incorporated into G-quadruplex DNA has revealed efficient energy transfer reactions in cation-containing G-quadruplexes. 8-(2-Pyridyl)-2'-deoxyguanosine, "2PyG", is a highly sensitive internal fluorescent probe of G-quadruplex folding and energy transfer. 2PyG was minimally disruptive to G-quadruplex folding and exhibited intense fluorescence, even when it was base-stacked with other guanine residues. Using 2PyG we have quantified energy transfer efficiencies within G-quadruplex structures prepared under conditions of excess Na(+)/K(+) (110 mM) or in 40% polyethylene glycol (PEG) under salt deficient conditions. G-quadruplex structures containing coordinated cations exhibited efficient DNA-to-probe energy transfer reactions (η(t) = 0.11-0.41), while PEG-folded G-quadruplexes exhibited very little energy transfer (η(t) = 0.02-0.07). Experiments conducted using unmodified G-quadruplexes suggest that cation coordination at the O(6) position of guanine residues results in enhanced quantum yields of G-quadruplex nucleobases that, in turn, serve as efficient energy donors to 2PyG. Given the growing interest in G-quadruplex-based devices and materials, these results will provide important design principles toward harnessing the potentially useful photophysical properties of G-quadruplex wires and other G-rich structures.     

Raman spectroscopic study of the conformational change of 12-crown-4 due to cation capture

Raman spectra of the Li⁺, Na⁺, K⁺, NH⁺₄, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Pb²⁺ complexes of 12-crown-4 and also 12-crown-4 in various states are observed. The spectra of 12-crown-4 change remarkably by complex formation with cations. Normal vibration calculations of various conformations of 12-crown-4 are carried out. On the basis of the observed spectra and the results of the calculations, the conformation of 12-crown-4 in the Li⁺, Na⁺, K⁺, NH⁺₄, Mg²⁺ complexes is found to have approximate D_2d symmetry, while that in the Ca²⁺, Sr²⁺, Ba²⁺ complexes is found to have approximate C_2V symmetry.     

A conformationally constrained nucleotide analogue controls the folding topology of a DNA g-quadruplex

Guanine-rich DNA and RNA sequences can fold into unique structures known as G-quadruplexes. The structures of G-quadruplexes can be divided into several classes, depending on the parallel or antiparallel nature of the strands and the number of G-rich tracts present in an oligonucleotide. Oligonucleotides with single tracts of guanines form intermolecular parallel tetrameric G-quadruplexes. Oligonucleotides with two tracts of guanosines separated by two or more bases can form both intermolecular antiparallel fold-back dimeric and parallel tetrameric G-quadruplexes, and those with four tracts of guanosines can form both intramolecular parallel and antiparallel structures. Intramolecular G-qaudruplexes can fold into several folding topologies including antiparallel crossover basket, antiparallel chair, and parallel propeller. The ability to control the folding of G-quadruplexes would allow the physical, biochemical, and biological properties of these various folding topologies to be studied. Previously, the known methods to control the folding topology of G-quadruplexes included changing the buffer by varying the mono- and divalent cations that are present, and by changing the DNA sequence. Because the glycosidic bonds in the G-quartets of G-quadruplexes with parallel strands are in the anti conformation, we reasoned that incorporation of nucleoside analogues that prefer the anti conformation of the glycosidic bond into G-rich sequences would increase the preference for parallel G-quadruplex formation. As predicted, by positioning the conformationally constrained nucleotide analogue 2'-O-4'-C-methylene-linked ribonucleotide into specific positions of a DNA G-quadruplex we were able to shift the thermodynamically favored structure of a G-quadruplex from an antiparallel to a parallel structure.     

Selection of G-quadruplex folding topology with LNA-modified human telomeric sequences in K+ solution

G-rich nucleic acid oligomers can form G-quadruplexes built by G-tetrads stacked upon each other. Depending on the nucleotide sequence, G-quadruplexes fold mainly with two topologies: parallel, in which all G-tracts are oriented parallel to each other, or antiparallel, in which one or more G-tracts are oriented antiparallel to the other G-tracts. In the former topology, all glycosidic bond angles conform to anti conformations, while in the latter topology they adopt both syn and anti conformations. It is of interest to understand the molecular forces that govern G-quadruplex folding. Here, we approach this problem by examining the impact of LNA (locked nucleic acid) modifications on the folding topology of the dimeric model system of the human telomere sequence. In solution, this DNA G-quadruplex forms a mixture of G-quadruplexes with antiparallel and parallel topologies. Using CD and NMR spectroscopies, we show that LNA incorporations can modulate this equilibrium in a rational manner and we establish a relationship between incorporation of LNA nucleotides in syn and/or anti positions and the shift of the equilibrium to obtain exclusively the parallel G-quadruplex. The change in topology is driven by a combination of the C3'-endo puckering of LNA nucleotides and their preference for the anti glycosidic conformation. In addition, the parallel LNA-modified G-quadruplexes are thermally stabilised by about 11 °C relative to their DNA counterparts.     

Pb2+和Zn2+对亲水型光敏热成像材料感光性能和存储稳定性能的影响

分别研究了Pb²⁺和Zn²⁺对以聚乙烯醇(PVA)为粘合剂的亲水型光敏热成像(PTG)材料的感光性能以及老化稳定性能的影响.实验结果表明,涂布液中加入Pb²⁺或Zn²⁺后,PTG材料的热显影灰雾和相对感光度均明显降低,并且随着离子加入量的增加,下降程度更加显著.另外,含有Pb²⁺或Zn²⁺的PTG材料在经过不同时间的老化后,与未老化PTG材料相比,其热显影灰雾缓慢增加,相对感光度也略有上升.     

Synthesis and fluorescence behaviour of crown and azacrown ethers carrying the dansyl fluorophore as a pendant in acetonitrile solution

The influence of Li⁺, Na⁺, K⁺, Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Pb²⁺ and Al³⁺ ions on the spectroscopic properties of the dansyl group covalently linked to crown ether or diazacrown ethers was investigated by means of absorption and emission spectrophotometry. Interaction of the alkali metal ions with all fluoroionophores studied is weak, while alkaline earth metal ions interact strongly causing about 50% quenching of dansyl fluorescence of A21C5-Dns and A₂18C6-Dns. The Cu²⁺, Pb²⁺ and Al³⁺ cations interact very strongly with dansyl chromophore regardless the crown ether type, causing a major change in absorption spectrum of the chromophore and forming non-fluorescent complexes. The Co²⁺, Ni²⁺, Zn², Mg²⁺ and Ag⁺ cations interact moderately with all fluoroionophores studied causing about 20% of fluorescence quenching of dansyl, except for a strong dansyl fluorescence quenching of 15C5-Dns by Co²⁺ ion. The quenching efficiency of didansylated fluoroionophores by the alkali metal ions and alkaline earth metal ions is weaker than monodansylated ones.     

热剥离法制备石墨烯纳米片对Pb2+和Cd2+的吸附

石墨粉氧化后,在氮气气氛下,快速高温剥离制得石墨烯纳米片。采用X射线衍射(XRD)、扫描电子显微镜(SEM)、透射电子显微镜(HRTEM)、拉曼(Raman)光谱、傅里叶变换红外(FT-IR)光谱和氮气吸附-脱附等分析手段对石墨烯样品进行了表征。这些分析测试结果显示:石墨烯样品主要由很薄的1-4层石墨组成,呈褶皱状态,比表面积为628.5 m²·g^-1。研究了石墨烯吸附水溶液中的Pb²⁺和Cd²⁺的pH值、吸附时间、吸附温度和金属离子初始浓度等影响因素, Pb²⁺和Cd²⁺的最大吸附量分别为460.20和72.39 mg·g^-1。结果表明,热剥离法制得的高质量石墨烯纳米片可以作为一种高效的从水中去除Pb²⁺和Cd²⁺的吸附材料。