基于对硫黄素T诱导的DNA G-四链体抑制作用检测银离子

发展了基于DNA探针的新型银离子免标记检测方法。富含C和G碱基的DNA序列可在荧光染料硫黄素T(Th T)的诱导下折叠形成四链体结构,并使Th T的荧光显著增强,但该Th T—DNA复合形成的四链体易被银离子破坏。依据此特性,设计了一种新型的免标记银离子传感器。银离子的响应范围为0.1~10μmol/L,检出限为60 nmol/L,具有较高的灵敏度。该方法已成功应用于湘江水和自来水样品中银离子的检测,结果表明Th T—DNA复合四链体有望提供一种可靠、高灵敏和特异的检测平台,实现复杂体系中银离子的定量分析。     

基于G-四链体/硫黄素T的荧光生物传感器检测Pb^(2+)北大核心CSCD

本研究利用Pb^(2+)与硫黄素T(ThT)对功能核酸G-四链体(G4)中心位点的竞争关系,构建了一种荧光生物传感器用于Pb^(2+)的简单灵敏检测。ThT可以特异性结合G4,且在结合后显示出明显的荧光信号。Pb^(2+)能与G4形成更稳定的结构,故而当溶液中存在Pb^(2+)时,ThT会从G4-ThT体系中被竞争释放出来,失去受G4束缚状态下的刚性结构,从而降低了其荧光强度。在最优条件下,该体系荧光信号与Pb^(2+)浓度在5~1000 nmol/L范围内呈现良好的线性关系,检测限为1.6 nmol/L,同时实现对中药材独活中Pb^(2+)含量的加标测定。方法具有操作简便、响应快速以及高选择性超灵敏的特点,在Pb^(2+)检测方面有良好的应用潜力。     

DNA G-四链体的结构完整性对催化性质的影响

DNA酶中的G-四链体-血红素(G4-hemin)DNA酶结构具有较高的设计性和化学稳定性,因此格外受研究者关注.G-平面作为辅酶因子hemin的结合位点,不仅提供大π平面与hemin结合,而且其平面上的G碱基还可以充当近端配位基团与hemin进行配位.因此,研究G-平面完整性在G4-DNA酶体系中的作用具有重要意义....     

钌配合物与G-四链体DNA的相互作用研究进展

人体端粒由富含鸟嘌呤(G)的DNA重复序列组成,该序列在一定条件下可以形成G-四链体DNA结构。小分子化合物诱导该结构的形成并使之稳定,可以抑制端粒酶活性而达到抗肿瘤的目的。因此,G-四链体DNA稳定剂的设计和筛选是近年来生物无机化学的重要前沿研究领域之一。在金属配合物中,钌配合物由于具有丰富的光化学、光物理特性以及生物活性,其作为G-四链体DNA稳定剂引起人们的高度关注。本文以近年一些代表性的研究工作为例,对钌配合物与G-四链体DNA相互作用方面的研究进展进行了综述。     

卟啉衍生物作为G-四链体DNA结合剂的研究进展

综述了卟啉衍生物作为G-四链体DNA结合剂的研究进展,其中涉及该类化合物作为G-四链体DNA结合剂的设计合成、亲和能力以及端粒酶抑制活性.     

类黄酮化合物对G-四链体及双链DNA选择性的电喷雾质谱研究

利用电喷雾质谱(ESI-MS)研究了4种常见的类黄酮化合物芦丁、 槲皮素、 葛根素和柚皮苷与2种不同形态结构的G-四链体DNA和3种双链DNA的非共价相互作用, 比较了这些小分子化合物与不同形态结构DNA结合的强弱及形成复合物的化学计量. 结果表明, 芦丁和槲皮素对G-四链体DNA具有一定的选择性, 同时它们对双链DNA的选择性也较高; 而葛根素和柚皮苷对G-四链体DNA仅显示了较低的选择性.     

G-四链体DNA稳定剂的研究进展

人体端粒由富含鸟嘌呤(G)的DNA重复序列组成,该序列在一定的条件下可以形成G-四链体DNA的结构.小分子化合物诱导该结构的形成并使之稳定,不但可以抑制端粒酶的活性或降低癌基因的转录表达而达到抗肿瘤的目的,还可以作为G-四链体DNA的探针,辅助G-四链体DNA生物功能的研究及与之相关疾病的诊断.因此,G-四链体DNA稳定剂的设计是近年来化学生物学的重要前沿领域之一.到目前为止,G-四链体DNA稳定剂主要可分为有机小分子化合物和金属配合物.本文重点综述这两方面特别是后者的最新研究进展.     

邻菲罗啉衍生物与G-四链体DNA的识别

设计合成了一种新型邻菲罗啉衍生物,用IR,<'1>H-NMR,<'13>C-NMR和质谱进行了结构表征.在包含100mM NaCl的Tris-HCl(pH 7.4)缓冲溶液中,利用荧光共振能量转移熔点(FRET-melting)分析以及紫外可见光谱研究了此化合物对人端粒G-四链体DNA(AG<,3>T<,2>AG<,3...     

DNA G-四链体识别探针研究进展

G-四链体是一种由富含鸟嘌呤核酸序列形成的独特的二级结构,广泛分布于真核生物基因组,如端粒DNA、r DNA和一系列基因中的启动子区域。G-四链体结构对很多重要的生理过程如基因的转录、复制、重组以及保持染色体的稳定性方面具有重要作用。G-四链体的特异、高灵敏检测将为进一步了解G-四链体结构在人类细胞基因组中的分布、功能和机制奠定基础,也可能为靶向G-四链体的肿瘤治疗方法提供新的思路。因而过去几十年人们一直致力于开发设计具有高选择性和高灵敏度的G-四链体识别探针,这些探针已经广泛应用于溶液中G-四链体的识别,而且具有良好的选择性。目前也有少数探针能够直接用于检测活体G-四链体结构。本文综述了一些常见的靶向G-四链体的小分子配体,以及它们在染色体和活体细胞G-四链体检测中的应用。笔者希冀本文能为设计识别G-四链体的高性能探针,进一步实现活细胞内G-四链体的检测提供借鉴。     

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.     

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^...     

Single-molecule investigation of human telomeric G-quadruplex interactions with Thioflavin T

The interactions between human telomere sequence and a typical highly selective G-quadruplex ligand ThT were studied at the single-molecule level through α-hemolysin protein nanopore.     

Flexible RuII Schiff Base Complexes: G-Quadruplex DNA Binding and Photo-Induced Cancer Cell Death

A series of new Ru^II Schiff base complexes built on the salphen moiety has been prepared. This includes four flexible monometallic Ru^II compounds and six rigid bimetallic analogues that contain Ni^II, Pd^II or Pt^II cations into the salphen complexation site. Steady state luminescence titrations illustrated the capacity of the compounds to photoprobe G-quadruplex (G4) DNA. Moreover, the vast array of the Schiff base structural changes allowed to extensively assess the influence of the ligand surface, flexibility and charge on the interaction of the compounds with G4 DNA. This was achieved thanks to circular dichroism melting assays and bio-layer interferometry studies that pointed up high affinities along with good selectivities of Ru^II Schiff base complexes for G4 DNA. In cellulo studies were carried out with the most promising compounds. Cellular uptake with location of the compounds in the nucleus as well as in the nucleolus was observed. Cell viability experiments were performed with U2OS osteosarcoma cells in the dark and under light irradiation which allowed the measurements of IC₅₀ values and photoindexes. They showed the substantial role played by light irradiation in the activity of the drugs in addition to the low cytotoxicity of the molecules in the dark. Altogether, the reported results emphasize the promising properties of Ru^II Schiff base complexes as a new class of candidates for developing potential G4 DNA targeting diagnostic or therapeutic compounds.     

基于"分段"-"完整"转化的G-四链体脱氧核糖核酸酶传感器用于多聚核苷酸激酶的检测

采用"分段"转化为"完整"G-四链体脱氧核糖核酸酶的策略,构建了检测多聚核苷激酶(T4 PNK)的传感器.将形成G-四链体的富G序列PS5.M序列拆分成5 '和3'端均为羟基的两条链:链S1OH和链S2OH,即分别具有12个碱基的"分段"的PS5.M.在ATP存在下,T4 PNK酶可以将链S2OH的5 '端的羟基磷酸化,转化为S2P;在S1OH、S2P以及Helper链S—H存在下,T4 DNA连接酶(T4 DNA Ligase)将链S1OH和链S2P连接成"完整"的PS5.M序列.在体系中再加入核酸外切酶Ⅲ(ExoⅢ),从S—H的3'端剪切S-H,释放出PS5.M.在K+存在下,PS5.M与氯化血红素(Hemin)作用,形成具有类过氧化物酶活性的复合物,催化H2O2氧化2,2'-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)的反应,通过检测氧化产物在418 nm处的吸收值变化,实现对T4 PNK活性的定量检测.线性检测范围为0.02 ～ 3.0 U/mL,检出限为0.014 U/mL(S/N=3).对Hela细胞和HEK293细胞实际样本的T4 PNK活性进行了检测,平均回收率为95.6％～105.7％.     

A Label-Free Fluorescence Aptasensor Based on G-Quadruplex/Thioflavin T Complex for the Detection of Trypsin

A novel, label-free fluorescent assay has been developed for the detection of trypsin by using thioflavin T as a fluorescent probe. A specific DNA aptamer can be combined by adding cytochrome c. Trypsin hydrolyzes the cytochrome c into small peptide fragments, exposing the G-quadruplex part of DNA aptamer, which has a high affinity for thioflavin T, which then enhances the fluorescence intensity. In the absence of trypsin, the fluorescence intensity was inhibited as the combination of cytochrome c and the DNA aptamer impeded thioflavin T’s binding. Thus, the fluorescent biosensor showed a linear relationship from 0.2 to 60 μg/mL with a detection limit of 0.2 μg/mL. Furthermore, the proposed method was also successfully employed for determining trypsin in biological samples. This method is simple, rapid, cheap, and selective and possesses great potential for the detection of trypsin in bioanalytical and biological samples and medical diagnoses.     

Using Macrocyclic G-Quadruplex Ligands to Decipher the Interactions Between Small Molecules and G-Quadruplex DNA

This study aims to deepen the knowledge of the current state of rational G4-ligand design through the design and synthesis of a novel set of compounds based on indoles, quinolines, and benzofurans and their comparisons with well-known G4-ligands. This resulted in novel synthetic methods and G4-ligands that bind and stabilize G4 DNA with high selectivity. Furthermore, the study corroborates previous studies on the design of G4-ligands and adds deeper explanations to why a) macrocycles offer advantages in terms of G4-binding and -selectivity, b) molecular pre-organization is of key importance in the development of strong novel binders, c) an electron-deficient aromatic core is essential to engage in strong arene-arene interactions with the G4-surface, and d) aliphatic amines can strengthen interactions indirectly through changing the arene electrostatic nature of the compound. Finally, fundamental physicochemical properties of selected G4-binders are evaluated, underscoring the complexity of aligning the properties required for efficient G4 binding and stabilization with feasible pharmacokinetic properties.     

Fluorescent dyes in the context of DNA‐binding: The case of Thioflavin T

Thioflavin T (ThT) is a dye characterized by a strong fluorescence light‐up on binding to biosubstrates. Although this effect is known to be related to the inhibition of intramolecular torsion on excitation, the binding modes and their role in affecting photoinduced processes are by no means adequately understood. Here, a combined molecular dynamics and quantum chemical modeling is used to study the tuning of the photophysical properties of ThT when moving from solution to DNA binding. The binding mechanism of ThT to B‐DNA was found to be very complex as a result of an uncommon interplay between different binding modes, for example, monomer intercalation and external binding but also groove binding of the dimer. The detailed analysis of the relation between the different binding modes and the structural and electronic properties of ThT can be used to better understand the interaction with other biosubstrates.     

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.