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.     

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.     

Mass spectrometric studies on effects of counter ions of TMPyP4 on binding to human telomeric DNA and RNA G-quadruplexes

A comparative study on human telomeric DNA G-quadruplex binding of meso-5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) between its two salt forms, i.e., tetratosylate and tetrachloride, was conducted by using ESI-TOF-MS, UV-melting measurement, and molecular modeling methods. Besides cation TMPyP4, the tosyl anion was found to bind to human telomeric DNA G-quadruplex with multiple binding stoichiometries from 1:1 to 3:1 observed in ESI-TOF-MS spectra, indicating that the stabilization activity of TMPyP4 tetratosylate on G-quadruplex is derived from a synergetic effect of both TMPyP4 cation and tosyl anion. A molecular modeling study suggests that a tosyl anion fills up the vacant space between TMPyP4 cation and DNA G-quadruplex and thus stabilizes the complex by 3.8 kcal/mol. Therefore, it is estimated that TMPyP4 tetratosylate’s activity might not reflect the real effect of TMPyP4 cation in some bioassays related to G-quadruplex stabilization. This was verified by the results of less binding affinity of TMPyP4 tetrachloride with DNA G-quadruplex obtained from ESI-TOF-MS measurement, and of 2.27 °C less thermal stabilization of TMPyP4 tetrachloride for DNA G-quadruplex, compared to its tetratosylate under the same conditions. Our study demonstrated the influence of counter ions of TMPyP4 on G-quadruplex binding, which sheds light on the proper usage of TMPyP4 salt in the chemical and biological research associated with G-quadruplex binding. Subsequently, the binding of TMPyP4 tetrachloride to human telomeric RNA G-quadruplexes was studied with ESI-TOF-MS technique. The binding constants of TMPyP4 with human telomeric G-quadruplexes indicated that TMPyP4 binds to human telomeric RNA G-quadruplex one order of magnitude stronger than DNA counterpart. This is a comprehensive mass spectrometric report on binding study of TMPyP4 with human telomeric DNA/RNA G-quadruplexes.

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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....     

Characterization of structure and stability of long telomeric DNA G-quadruplexes

In the current study, we used a combination of gel electrophoresis, circular dichroism, and UV melting analysis to investigate the structure and stability of G-quadruplexes formed by long telomeric DNAs from Oxytricha and human, where the length of the repeat (n)=4 to 12. We found that the Oxytricha telomeric DNAs, which have the sequence (TTTTGGGG)n, folded into intramolecular and intermolecular G-quadruplexes depending on the ionic conditions, whereas human telomeric DNAs, which have the sequence (TTAGGG)n, formed only intramolecular G-quadruplexes in all the tested conditions. We further estimated the thermodynamic parameters of the intramolecular G-quadruplex. We found that thermodynamic stabilities of G-quadruplex structures of long telomeric DNAs (n=5 to 12) are mostly independent of sequence length, although telomeric DNAs are more stable when n=4 than when n>or=5. Most importantly, when n is a multiple of four, the change in enthalpy and entropy for G-quadruplex formation increased gradually, demonstrating that the individual G-quadruplex units are composed of four repeats and that the individual units do not interact. Therefore, we propose that the G-quadruplexes formed by long telomeric DNAs (n>or=8) are bead-on-a-string structures in which the G-quadruplex units are connected by one TTTT (Oxytricha) or TTA (human) linker. These results should be useful for understanding the structure and function of telomeres and for developing improved therapeutic agents targeting telomeric DNAs.     

FeII4L4 tetrahedron binds and aggregates DNA G-quadruplexes

Since the discovery of the G-quadruplex (G4) structure in telomeres in 1980s, studies have established the role it plays in various biological processes. Here we report binding between DNA G4 and a self-assembled tetrahedral metal-organic cage 1 and consequent formation of aggregates, whereby the cage protects the DNA G4 from cleavage by S1 nuclease. We monitor DNA–cage interaction using fluorescence spectroscopy, firstly by quenching of a fluorescent label appended to the 5′ end of G4. Secondly, we detect the decrease in fluorescence of the G4-selective dyes thioflavin-T and Zn-PPIX bound to various DNA G4 sequences following the addition of cage 1. Our results demonstrate that 1 interacts with a wide range of G4s. Moreover, gel electrophoresis, circular dichroism and dynamic light scattering measurements establish the binding of 1 to G4 and indicate the formation of aggregate structures. Finally, we find that DNA G4 contained in an aggregate of cage 1 is protected from cleavage by S1 nuclease.

We find Fe^II₄L₄ binds to G-quadruplex and forms aggregates. G-quadruplex in the aggregates is protected from digestion by S₁ nuclease.     

Synthesis and characterization of a fluorescent adenosine derivative for detection of intermolecular RNA G-quadruplexes

We synthesized a fluorescent adenosine derivative, rA^py, as a probe to study RNA structural transitions, in particular the intermolecular G-quadruplex formation. rA^py was incorporated into the dangling positions of guanine-rich oligonucleotides, which under physiological conditions undergo π-stacking on top of each other exhibiting a strong emission signal in their G-quadruplex conformation, but not in their single-stranded state.     

The therapeutic application of lanthanides

The biological properties of the lanthanides, based on their similarity to calcium, have stimulated research into their therapeutic application. Historical medical uses of the lanthanides and recent advances and successes will be described in the context of the biological chemistry of lanthanides, including a new metal-based drug, lanthanum carbonate, which has recently been approved as a phosphate binder for the treatment of hyperphosphatemia. This tutorial review will be of interest to those working on metal-based drugs, including inorganic chemists, and biological scientists.     

Kinetically grafting G-quadruplexes onto DNA nanostructures for structure and function encoding via a DNA machine

Kinetically grafting G-quadruplexes onto one-dimensional DNA nanostructures with precise positioning was realized in this study. The programs hold great promise for label-free and enzyme-free detection of various targets as a result of signal amplification from G-quadruplexes, and building DNA nanostructures as scaffolds due to the molecular recognition capacity of G-quadruplex aptamers.     

DNA and RNA aptamers: from tools for basic research towards therapeutic applications

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.     

Synthesis and binding studies of novel diethynyl-pyridine amides with genomic promoter DNA G-quadruplexes

Herein, we report the design, synthesis and biophysical evaluation of novel 1,2,3-triazole-linked diethynyl-pyridine amides and trisubstituted diethynyl-pyridine amides as promising G-quadruplex binding ligands. We have used a Cu(I)-catalysed azide-alkyne cycloaddition click reaction to prepare the 1,2,3-triazole-linked diethynyl-pyridine amides. The G-quadruplex DNA binding properties of the ligands have been examined by using a F?rster resonance energy transfer (FRET) melting assay and surface plasmon resonance (SPR) experiments. The investigated compounds are conformationally flexible, having free rotation around the triple bond, and exhibit enhanced G-quadruplex binding stabilisation and specificity between intramolecular promoter G-quadruplex DNA motifs compared to the first generation of diaryl-ethynyl amides (J. Am. Chem. Soc. 2008, 130, 15950-15956). The ligands show versatility in molecular recognition and promising G-quadruplex discrimination with 2-50-fold selectivity exhibited between different intramolecular promoter G-quadruplexes. Circular dichroism (CD) spectroscopic analysis suggested that at higher concentration these ligands disrupt the c-kit2 G-quadruplex structure. The studies validate the design concept of the 1,3-diethynyl-pyridine-based scaffold and demonstrate that these ligands exhibit not only significant selectivity over duplex DNA but also variation in G-quadruplex interaction properties based on small chemical changes in the scaffold, leading to unprecedented differential recognition of different DNA G-quadruplex sequences.     

Diarylethynyl amides that recognize the parallel conformation of genomic promoter DNA G-quadruplexes

We report bis-phenylethynyl amide derivatives as a potent G-quadruplex binding small molecule scaffold. The amide derivatives were efficiently prepared in 3 steps by employing Sonogashira coupling, ester hydrolysis and a chemoselective amide coupling. Ligand-quadruplex recognition has been evaluated using a fluorescence resonance energy transfer (FRET) melting assay, surface plasmon resonance (SPR), circular dichroism (CD) and (1)H nuclear magnetic resonance (NMR) spectroscopy. While most of the G-quadruplex ligands reported so far comprise a planar, aromatic core designed to stack on the terminal tetrads of a G-quadruplex, these compounds are neither polycyclic, nor macrocyclic and have free rotation around the triple bond enabling conformational flexibility. Such molecules show very good binding affinity, excellent quadruplex:duplex selectivity and also promising discrimination between intramolecular promoter quadruplexes. Our results indicate that the recognition of the c-kit2 quadruplex by these ligands is achieved through groove binding, which favors the formation of a parallel conformation.     

Recent developments in novel blue/green/red/NIR small fluorescent probes for in cellulo tracking of RNA/DNA G-quadruplexes

The detection and stabilization of G-quadruplexes (G4s) in living systems is of enormous applicability in the fields of chemical biology and therapeutic materials. Whereas DNA serves as a genetic material, RNA functions in the regulation and expression of genetic materials. Even there is various report on fluorescent probes invitro G4s recognitions, in this review we highlighted briefly, in-cellulo identification of G4s along with conventional methods principles. Although there are varieties of G4-forming sequences in the genome, targeting a specific type (topology) in living cells is highly challenging because of the high instability of G4s in cellular/subcellular systems. In contrast, several reports describe the in vitro identification of G4s, along with in-cell demonstrations, using efficient fluorescent probes, through either intrinsic or extrinsic approaches. In the intrinsic mode, the sensing results from the use of highly selective synthetic fluorescent oligonucleotides or proteins (a labeling approach). In the extrinsic mode, quencher-free small molecular probes are used to recognize specific G4s under physiological conditions. Because of their robustness, simplicity, and ease of handling, this review describes recent trends in the use of blue/green, green, red, and near-infrared (NIR) fluorescent probes for the recognition of G4s in live cells-and, particularly, those approaches employing quencher-free probes. Also highlighted are a few labeled probes, and their in cellulo localizations, which were accomplished upon the formation of non-canonical G4s under specified conditions and supplemented by exogenous G4-forming components, without harnessing cellular physiological conditions.     

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。     

Multiple chromatographic fingerprinting and its application to the quality control of herbal medicines

Recently, chromatographic fingerprinting has become one of the most powerful approaches to quality control of herbal medicines. However, the performance of reported chromatographic fingerprinting constructed by single chromatogram sometimes turns out to be inadequate for complex herbal medicines, such as multi-herb botanical drug products. In this study, multiple chromatographic fingerprinting, which consists of more than one chromatographic fingerprint and represents the whole characteristics of chemical constitutions of the complex medicine, is proposed as a potential strategy in this complicated case. As a typical example, a binary chromatographic fingerprinting of “Danshen Dropping Pill” (DSDP), the best-sold traditional Chinese medicine in China, was developed. First, two HPLC fingerprints that, respectively, represent chemical characteristics of depsides and saponins of DSDP were developed, which were used to construct binary chromatographic fingerprints of DSDP. Moreover, the authentication and validation of the binary fingerprints were performed. Then, a data-level information fusion method was employed to capture the chemical information encoded in two chromatographic fingerprints. Based on the fusion results, the lot-to-lot consistency and frauds can be determined either using similarity measure or by chemometrics approach. The application of binary chromatographic fingerprinting to consistency assessment and frauds detection of DSDP clearly demonstrated that the proposed method was a powerful approach to quality control of complex herbal medicines.     

Spectral correlative chromatography and its application to analysis of chromatographic fingerprints of herbal medicines

A signal-processing method known as spectral correlative chromatography (SCC) for two-dimensional data obtained from hyphenated chromatography is developed and applied to chemical chromatographic fingerprint data sets of herbal medicine under specific experimental conditions. The method can judge the presence or absence of a spectral correlative peak among the spectrochromatograms. A local least squares regression model (LLS) is constructed in a piecewise manner to correct the shifts of retention time of some peaks of interest in the chromatograms of various test samples. The results compare favorably with those obtained by a two-point calibrated algorithm. It is shown that performing SCC and LLS on the piecewise clusters of various chromatographic fingerprints is more helpful in practice in revealing their common nature and for characterizing the chemical constituents. This approach holds great potential for facilitating quality control of herbal medicines.     

Redox cycling amplified electrochemical detection of DNA hybridization: application to pathogen E. coli bacterial RNA

An electrochemical genosensor in which signal amplification is achieved using p-aminophenol (p-AP) redox cycling by nicotinamide adenine dinucleotide (NADH) is presented. An immobilized thiolated capture probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the detection probe, and streptavidin-alkaline phosphatase as reporter enzyme. The phosphatase liberates the electrochemical mediator p-AP from its electrically inactive phosphate derivative. This generated p-AP is electrooxidized at an Au electrode modified self-assembled monolayer to p-quinone imine (p-QI). In the presence of NADH, p-QI is reduced back to p-AP, which can be re-oxidized on the electrode and produce amplified signal. A detection limit of 1 pM DNA target is offered by this simple one-electrode, one-enzyme format redox cycling strategy. The redox cycling design is applied successfully to the monitoring of the 16S rRNA of E. coli pathogenic bacteria, and provides a detection limit of 250 CFU μL⁻¹.     

Tetra-end-linked oligonucleotides forming DNA G-quadruplexes: a new class of aptamers showing anti-HIV activity

The biophysical and biological properties of unprecedented anti-HIV aptamers are presented. The most active aptamer (1L) shows a significant affinity to the HIV protein gp120.