G-四链体的构型、热稳定性测试及其在钾离子定量检测中的应用

G-四链体是由具有连续鸟嘌呤(G)序列的DNA或RNA形成的一种特殊的核酸二级结构,由于有望形成G-四链体结构的序列广泛地分布于人类基因组的许多重要区域,有关G-四链体的研究已经成为国际上的一个研究热点。本文对G-四链体构型的多态性、G-四链体热稳定性的测试手段及G-四链体在K+定量检测方面的应用研究进行了简单的介绍和评述。     

基于G-四链体-血红素和链式放大反应的基因检测传感器的构建

该文以特殊设计的DNA序列为捕获探针,以G-四链体-血红素复合物作为信号分子,利用链式反应实现目标DNA的灵敏检测。在目标DNA存在时,捕获探针与目标DNA相互识别,同时目标DNA能与辅助探针发生连续的链式反应,从而在电极表面引入大量G-四链体结构。血红素存在下,G-四链体可与血红素结合形成具有很强电化学信号的G-四链体-血红素复合物。用差分脉冲伏安法(DPV)扫描得到的电化学信号与体系中的目标DNA浓度存在对应关系,从而实现对目标DNA的检测。在各组分浓度最适的情况下,电流响应值与目标DNA浓度在0.01～10 pmol/L内具有良好的线性关系,检出限可达8 fmol/L。该传感器灵敏度高、特异性好,具有良好的应用前景。     

核酸G-四链体的识别、复合物结构与细胞内探测的研究进展

富含鸟嘌呤的DNA或RNA序列可以折叠成非典型G-四链体二级结构. G-四链体结构丰富多样,在生物体内动态存在,参与了转录、复制、基因组稳定性和表观遗传调控等关键的基因组功能,与癌症生物学密切相关. G-四链体的结构与功能机制研究促进了以G-四链体为靶点的肿瘤治疗干预.本文综合评述了核酸G-四链体的特异性识别、细胞内探测及生物学功能的调控,总结了识别靶向G-四链体的小分子及复合物结构的研究进展,讨论了以G-四链体为靶点的靶向干预及疾病治疗的可能性,最后展望了G-四链体未来研究所面临的挑战与机遇.     

靶向G-四链体的小分子配体在癌症治疗中应用的研究进展

G-四链体是一类由Hoogsteen氢键维持稳定的,富含鸟嘌呤的DNA或RNA二级结构。人类基因组中存在大量潜在的形成G-四链体的序列,所形成的G-四链体结构能够调控基因组的稳定性、DNA复制和基因表达,其中包括很多与癌症相关基因。因此寻找能够诱导DNA的G富集区域形成G-四链体结构的配体,进而筛选潜在抗癌药物的先导化合物,已成为癌症治疗研究的热点之一。本文对近年来发现和设计的以G-四链体为靶点的小分子配体,按照靶向的G-四链体结构类型和配体的分子结构进行分类,综述了这类化合物在癌症治疗方面的研究进展,分析了相关靶向治疗存在的问题,并对未来的研究方向进行了展望。     

人类端粒DNA的分子识别及其作用机制探讨

核酸中富含短的G-碱基重复的序列可以形成一种复杂的高级结构,称为G-四链体（G-quadruplex）.在基因组中,借助生物信息学发现这类富G序列广泛分布在基因的启动子区,特别是那些参与到复制中去的基因,例如癌基因.同时发现这类序列在mRNA的5′非翻译区（5′UTR）也广泛存在.这类序列在染色体末段端粒部位的存在及功能已得到充分阐明.已知端粒富含G-碱基序列,其3′末端以单链状态存在,这使得在一些小分子的选择性作用下端粒序列很容易形成G-四链体结构,进而破坏端粒结构,影响端粒酶活性.已知端粒酶在超过85%的肿瘤中过量表达,因此,端粒酶已经成为抗癌药物设计的特殊靶点,是目前本领域的研究热点之一.已发现系列配体通过有效抑制端粒酶而表现高的抗肿瘤活性.本文主要综述了近年来端粒G-四链体分子识别及其药物靶向的最新进展,并对其作用机理做了进一步的分析和探讨.     

选择性识别平行性G-四联体的方酸类近红外荧光探针的研究

G-四联体是抗肿瘤药物筛选的一个重要靶点.开发针对某些拓扑结构的G-四联体荧光探针对于研究其结构和生物学功能具有十分重要的意义.设计、合成了四个方酸类花菁荧光染料即CSTS,CSBE,CSEM和CSBM,并检测了其对不同类型DNA的选择性识别作用.结果表明,所合成的四个化合物在缓冲溶液中几乎没有荧光发射,加入正平行G-四联体之后荧光增强大约1 000倍;而加入反平行G-四链体或单双链DNA荧光仅仅增强几十倍,说明其可以特异性识别平行G-四联体.但流式实验结果显示,CSTS不能透过细胞膜,同时存在高荧光背景的缺点,因此无法应用于活体分析.而另外三个不带阴离子侧链的衍生物则容易进入细胞,进入细胞的难易顺序为CSBECSEMCSBMCSTS.高选择性、低背景荧光和易进入细胞等优点使CSBE具有作为近红外荧光探针检测生物样品中正平行G-四联体的潜力.     

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。     

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...     

用于监测G-四联体结构变化及K^＋定量检测的荧光探针研究

有望形成G-四联体的基因序列广泛分布于真核细胞基因组中的许多重要区域。考虑到某些基因的生理功能可能与其采取的G-四联体结构密切相关,因此发展一种G-四联体结构探针用以区分不同结构的G-四联体就显得尤为必要。结晶紫是一种三苯甲烷类染料,其自身的荧光强度很弱,但当其与G-四联体结合后荧光强度大幅提升。究其原因,除了G-四联体与结晶紫的结合作用限制了染料分子的自由运动,使分子的平面性增强以外,G-四联体的环部结构对结合在G-四联体末端的染料分子的保护作用也是一个重要因素。     

原癌基因c-myc G-四链体配体甲基蓝衍生物的设计、合成及其活性研究

采用计算机辅助药物设计方法,将以甲基蓝为先导化合物设计的配体分子与端粒DNA、原癌基因cmyc、c-kit2等形成的G-四链体三维结构进行分子对接模拟,发现目标化合物选择性靶向c-myc G-四链体,其对接分值为7.74。以吩噻嗪为起始原料合成出目标化合物,其结构经~1H-NMR、~(13)C-NMR和HRMS等确证。采用圆二色光谱实验测试了化合物与端粒、原癌基因c-myc和c-kit2等DNA的相互作用,结果表明目标化合物选择性诱导c-myc DNA形成G-四链体。     

Quantitative Detection of G-Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination

G-quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G-quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE , which showed distinguishable fluorescence lifetime responses between G-quadruplexes and other DNA topologies, and fluorescence quantum yield (Φ_f) enhancement upon G-quadruplex binding. We determined two NBTE -G-quadruplex complex structures with high Φ_f values by NMR spectroscopy. The structures indicated NBTE interacted with G-quadruplexes using three arms through π–π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φ_f values and lifetime response of NBTE upon G-quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G-quadruplex DNA in live cells with NBTE and found G-quadruplex DNA content in cancer cells is 4-fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.     

Ligand Design to Acquire Specificity to Intended G-Quadruplex Structures

A G-quadruplex is a nucleic acid secondary structure that is adopted by guanine-rich sequences, and is considered to be relevant in various pharmacological and biological contexts. G-Quadruplexes have also attracted great attention in the field of DNA nanotechnology because of their extremely high thermal stability and the availability of many defined structures. To date, a large repertory of DNA/RNA G-quadruplex-interactive ligands has been developed by numerous laboratories. Several relevant reviews have also been published that have helped researchers to grasp the full scope of G-quadruplex research from its outset to the present. This review focuses on the G-quadruplex ligands that allow targeting of specific G-quadruplexes. Moreover, unique ligands, successful methodologies, and future perspectives in relation to specific G-quadruplex recognition are also addressed.     

Fluorescent probes for the stabilization and detection of G-quadruplexes and their prospective applications

This review article discusses the non-covalent interaction of various probe molecules of different structural diversity with G-quadruplex forming Guanine rich DNA sequences, revealing remarkable stimuli responsive fluorescence changes which are appropriate for sensing and other technological applications. Tailor-made probes having quadruplex inducing/stabilizing attachments of well-known dye molecules and its derivatives such as, coumarins, cyanines, thiazole orange, pyrazines, thioflavin T, triphenylmethane, tetraphenylethene, dimethylindole red etc have been employed. These probes express their modulations due to the binding to the topologically distinct G-quadruplexes through structural rigidization, aggregation propensity, binding strengths, tunability and other competitive interactions and are viewed as remarkable changes particularly in their fluorescence features. Based on this concept, several studies have reported the development of label free fluorescence sensor for the selective detection of topology specific G-quadruplexes, therapeutic, early diagnostic of cancer, cation-sensing, trace level detection of an anti-cancer drugs etc. have been accomplished. In vivo imaging is also achieved using a cholesterol attached G-quadruplex forming oligonucleotide probe labelled with specific dyes. Since the fine details of the topological information and control mechanisms of G-quadruplex forming sequences are very much essential for targeting and tuning several important biological processes relevant to cancer proliferation and developing stimuli responsive sensors, it is sure that many more contributions in this field will emerge in the coming years.     

Analytical potential of the quadruplex DNA-based FRET probes

DNA exhibits structural flexibility and may adopt also tetraplex structures known as guanine-quadruplexes or G-quadruplexes. These G-quadruplexes have recently received great attention because G-rich sequences are often found in genome and because of their potential links to mechanisms that relate to cancer, HIV, and other diseases. The unique structure of quadruplexes has also stimulated development of new analytical and bioanalytical assays based on fluorescence resonance energy transfer (FRET). Intramolecular folding of a flexible single-stranded DNA molecule into a compact G-quadruplex is a structural transition leading to closer proximity of its 5'- and 3'-ends. Thus, labeling both ends of a DNA strand with donor and acceptor fluorophores enables monitoring the quadruplex formation process by means of the FRET signal. This review shows how FRET technique contributes to G-quadruplex research and focuses mainly on analytical applications of FRET-labeled quadruplexes. Applications include studies of structural transitions of quadruplexes, FRET-based selection of ligands that bind to quadruplexes, design of molecular probes for protein recognition and development of sensors for detection of potassium ions in aqueous solution.     

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).     

Natural products and their derivatives as G-quadruplex binding ligands

G-quadruplexes comprise a class of secondary structures that are formed in guanine-rich sequences in eukaryotic genomes and play a crucial role in the regulation of many biological events. G-quadruplexes have become targets for anticancer drugs with high selectivity vs. duplex DNA and low cytotoxicity against normal cells. Natural products and their derivatives display polymorphism, structural complexity, and potent activity. It is, therefore, reasonable to seek ligands targeting G-quadruplexes from natural products. Recently, many successful examples have been reported, showing ligands with excellent anticancer activities. In this review, we summarized the development of research on natural products and derivatives that target G-quadruplex structures in an effort to guide future studies.     

Parallel G-quadruplex Structures Increase Cellular Uptake and Cytotoxicity of 5-Fluoro-2′-deoxyuridine Oligomers in 5-Fluorouracil Resistant Cells

Fluoropyrimidines, such as 5-fluorouracil (5-FU) and related prodrugs have been considered first-line chemotherapy agents for the treatment of colorectal cancer. However, poor specificity and tumor cell resistance remain major limiting bottlenecks. G-quadruplexes, have been suggested as preferred nanostructures for enhancing cellular uptake mediated by G-quadruplex binding proteins which are abundant at the membranes of some tumor cells. In the current study, we propose a new strategy to deliver 5-fluoro-2′-deoxyuridine (5-FdU) monophosphate, the main active drug from 5-FU derivatives that may circumvent the cellular mechanisms of FU-resistant cancer cells. Two G-quadruplexes delivery systems containing four and six G-tetrads ((TG₄T) and (TG₆T)) linked to a FdU oligonucleotide were synthesized. Biophysical studies show that the G-quadruplex parallel structures are not affected by the incorporation of the 5 units of FdU at the 5’-end. Internalization studies confirmed the ability of such G-quadruplex nanostructures to facilitate the transport of the FdU pentamer and increase its cytotoxic effect relative to conventional FU drug in FU-resistant colorectal cancer cells. These results suggest that FdU oligomers linked to G-quadruplex parallel sequences may be a promising strategy to deliver fluoropyrimidines to cancer cells.     

Phthalocyanines: a new class of G-quadruplex-ligands with many potential applications

A G-quadruplex is a four-stranded DNA structure featuring stacked guanine tetrads, G-quartets. Formation of a G-quadruplex in telomere DNA can inhibit telomerase activity; therefore, development of G-quadruplex-ligands, which induce and/or stabilize G-quadruplexes, has become an area of great interest. Phthalocyanine derivatives have substantial potential as high-affinity G-quadruplex-ligands because these planar chromophores are similar in size and shape to the G-quartets. Here, we focus on the latest findings on phthalocyanine derivatives as G-quadruplex-ligands, and discuss the mechanisms by which phthalocyanines bind to G-quadruplexes with high affinity and selectivity. We also discuss potential biomedical and organic electronic applications of phthalocyanines that are dependent on their photophysical properties.     

Specific Binding of Anionic Porphyrin and Phthalocyanine to the G-Quadruplex with a Variety of <em>i</em><em>n Vitro</em> and<em> </em><em>i</em><em>n Vivo</em> Applications

The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles in vitro and in vivo. In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin). Importantly, hemin binds to G-quadruplexes with high selectivity over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is attributable to an electrostatic repulsion of phosphate groups in ssDNA and dsDNA. The G-quadruplex and hemin-G-quadruplex complex allow development of sensing techniques to detect DNA, metal ions and proteins. In addition to hemin, anionic phthalocyanines also bind to the G-quadruplex formed by human telomere DNA, specifically over ssDNA and dsDNA. Since the binding of anionic phthalocyanines to the G-quadruplex causes an inhibition of telomerase activity, which plays a role in the immortal growth of cancer cells, anionic phthalocyanines are promising as novel anticancer drug candidates. This review focuses on the specific binding of hemin and anionic phthalocyanines to G-quadruplexes and the applications in vitro and in vivo of this binding property.