Regulation and recognization of the extended G-quadruplex by rutin

Rutins, a kind of flavonoid glycoside, showed different absorption and fluorescence spectral characteristics when bound to blunt-end stacked and interlocked extended G-quadruplexes. Further (1)H NMR spectra showed these two extended G-quadruplexes bound with rutins in different manners: after binding, the blunt-end stacked extended G-quadruplexes were decomposed to monomers, and the interlocked extended G-quadruplexes did not change. Based on these spectral features, one may expect the usage of rutins in recognizing different structural extended G-quadruplexes and regulating the ratio between blunt-end stacked extended G-quadruplexes and monomers.     

A new cationic porphyrin derivative (TMPipEOPP) with large side arm substituents: a highly selective G-quadruplex optical probe

The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA) sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4), interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1-piperidinyl)ethoxy]phenyl} porphyrin (TMPipEOPP), with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.     

Verification of specific G-quadruplex structure by using a novel cyanine dye supramolecular assembly: II. The binding characterization with specific intramolecular G-quadruplex and the recognizing mechanism

The supramolecular assembly of a novel cyanine dye, 3,3′-di(3-sulfopropyl)-4,5,4′,5′-dibenzo-9-ethyl-thiacarbocyanine triethylammonium salt (ETC) was designed to verify specific intramolecular G-quadruplexes from duplex and single-strand DNAs. Spectral results have shown that ETC presented two major distinct signatures with specific intramolecular G-quadruplexes in vitro: (i) dramatic changes in the absorption spectra (including disappearance of absorption peak around 660 nm and appearance of independent new peak around 584 nm); (ii) ∼70 times enhancement of fluorescence signal at 600 nm. Furthermore, based on ¹H-nuclear magnetic resonance and circular dichroism results, the preferring binding of ETC to specific intramolecular G-quadruplexes probably result from end-stacking, and the loop structure nearby also plays an important role.     

Studying the effect of crowding and dehydration on DNA G-quadruplexes

Intracellular environment is crowded with biomolecules that occupy a significant fraction (up to 40%) of the cellular volume, with a total concentration in the range 300-400mg/ml. Recently, the effect of crowding/dehydrating agents on the DNA G-quadruplexes has become a subject of an increasing interest. Crowding and/or dehydrating agents have been used to simulate how G-quadruplexes behave under cell-mimicking conditions characterized by a large excluded volume and a lower water activity. Indeed, the presence of both steric crowding and a lower water activity can affect G-quadruplex stability, their folding/unfolding kinetics, as well as their binding processes with proteins or small ligands. Many of these effects can be explored experimentally by measuring the dependence of the conformational stability, isomerisation kinetics and equilibria on the concentration of cosolutes which do not interact with the molecules (G-quadruplexes) under investigation. Spectroscopic methodologies, like circular dichroism, UV and fluorescence, have been widely employed to study G-quadruplexes in dilute solution. Here we focus on some aspects that need to be taken into account when employing such techniques in the presence of large amount of a cosolute. Additionally, we discuss possible problems/artifacts that arise in setting experiments in presence of these commonly employed cosolutes and in interpreting the results.     

Spectroscopic studies of the interaction between quercetin and G-quadruplex DNA

Quercetin is a kind of flavonoid which has been proved to exhibit anti-tumor activity. The interaction modes of quercetins with monomeric and dimeric G-quadruplexes were studied by absorption, fluorescence, CD, and (1)H NMR spectroscopies. The ligands were found to be stacked with terminal tetrads of monomeric G-quadruplexes by intercalation and bound to dimeric G-quadruplexes by groove binding.     

A spectroscopic study on the interactions of porphyrin with G-quadruplex DNAs

Free-base porphyrin (5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H,23H-porphine) (H(2)TMPyP4) has been shown to be an effective telomerase inhibitor by an in vitro assay. Here, we examined the interactions of the H(2)TMPyP4 with three distinct G-quadruplex DNAs, the parallel-stranded (TG(4)T)4, dimer-hairpin-folded (G(4)T(4)G(4))2, and monomer-folded AG(3)(T(2)AG(3))(3), by ultraviolet resonance Raman spectroscopy (UVRR), UV-vis absorption spectroscopy, fluorescence spectroscopy, and surface-enhanced Raman spectroscopy (SERS). The data obtained by the continuous variation titration method show that the binding stoichiometry of H(2)TMPyP4/G-quadruplex is 2:1 for (TG(4)T)4 and 4:1 for (G(4)T(4)G(4))2 or AG(3)(T(2)AG(3))(3). The results of SERS spectra, UV-vis absorption titration, and fluorescence emission spectra together with the binding stoichiometries reveal that two H(2)TMPyP4 molecules are externally stacked at two ends of the parallel (TG(4)T)4 G-quadruplex, whereas H(2)TMPyP4 molecules can intercalate within their diagonal or lateral loop regions and intervals between two G-tetrads for (G(4)T(4)G(4))2 and AG(3)(T(2)AG(3))(3) G-quadruplexes. The binding of H(2)TMPyP4 to (TG(4)T)4 G-quadruplex results in the hypochromicity of the UV Raman signal of (TG(4)T)4, indicating that the stacking effects between H(2)TMPyP4 and DNA bases are significant. The Raman hyperchromicities and shifts are observed after the binding of H(2)TMPyP4 to both (G(4)T(4)G(4))2 and AG(3)(T(2)AG(3))(3) G-quadruplexes. This indicates that the intercalative H(2)TMPyP4 can lengthen the vertical distance between adjacent G-tetrads of (G(4)T(4)G(4))2 and AG(3)(T(2)AG(3))(3) and change their conformations. The present study provides new insights into the effect of H(2)TMPyP4 binding on the structures of G-quadruplexes and also demonstrates that Raman spectroscopy is an ideal method for examining the interaction between drugs and G-quadruplexes.     

Emulsified BMVC derivative induced filtration for G-quadruplex DNA structural separation

A novel method based on emulsion/filtration is introduced for G-quadruplex DNA structural separation. We first synthesized a lipophilic analogue of BMVC, 3,6-Bis(1-methyl-4-vinylpyridinium)-9-(12′-bromododecyl) carbazole diiodide (BMVC-12C-Br), which can form an oil-in-water (o/w) phase emulsion. Due to the binding preferences of BMVC-12C-Br emulsion to some specific DNA structures, the large emulsion (∼2 µm) bound DNA was separated from the small free DNA in the filtrate by a 0.22 µm pore size MCE membrane. This method is able to isolate the non-parallel G-quadruplexes from the parallel G-quadruplexes and the linear duplexes from both G-quadruplexes. In addition, this method allows us not only to determine the absence of the parallel G-quadruplexes of d(T₂AG₃)₄ and the presence of the parallel G-quadruplexes of d(T₂AG₃)₂ in K⁺ solution, but also to verify structural conversion from antiparallel to parallel G-quadruplexes of d[AG₃(T₂AG₃)₃] in K⁺ solution under molecular PEG condition. Moreover, this emulsion can separate the non-parallel G-quadruplexes of d(G₃CGCG₃AGGAAG₅CG₃) monomer from the parallel G-quadruplexes of its dimer in K⁺ solution. Together with NMR spectra, one can simplify the spectra for both the free DNA and the bound DNA to establish a spectrum-structure correlation for further structural analysis.     

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three‐dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waal's forces are the main forces between pepsin and acteoside. According to the theory of Förster's non‐radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside. Copyright © 2015 John Wiley & Sons, Ltd.     

Measured and calculated CD spectra of G-quartets stacked with the same or opposite polarities

Circular dichroism (CD) spectroscopy is widely used to characterize the structures of DNA G-quadruplexes. CD bands at 200-300 nm have been empirically related to G-quadruplexes having parallel or antiparallel sugar-phosphate backbones. We propose that a more fundamental interpretation of the origin of the CD bands is in the stacking interactions of neighboring G-quartets, which can have the same or opposing polarities of hydrogen bond acceptors and donors. From an empirical summation of CD spectra of the d(G)5 G-quadruplex and of the thrombin binding aptamer that have neighboring G-quartets with the same and opposite polarities, respectively, the spectra of aptamers selected by the Ff gene 5 protein (g5p) appear to arise from a combination of the two types of polarities of neighboring G-quartets. The aptamer CD spectra resemble the spectrum of d(G3T4G3), in which two adjacent quartets have the same and two have opposite polarities. Quantum-chemical spectral calculations were performed using a matrix method, based on guanine chromophores oriented as in d(G3T4G3). The calculations show that the two types of G-quartet stacks have CD spectra with features resembling experimental spectra of the corresponding types of G-quadruplexes.     

Use of fluorescence spectroscopy to differentiate yeast and bacterial cells

This study focuses on the characterization of bacterial and yeast species through their autofluorescence spectra. Lactic acid bacteria (Lactobacillus sp.), and yeast (Saccharomyces sp.) were cultured under controlled conditions and studied for variations in their autofluorescence, particularly in the area representative of tryptophan residues of proteins. The emission and excitation spectra clearly reveal that bacterial and yeast species can be differentiated by their intrinsic fluorescence with UV excitation. The possibility of differentiation between different strains of Saccharomyces yeast was also studied, with clear differences observed for selected strains. The study shows that fluorescence can be successfully used to differentiate between yeast and bacteria and between different yeast species, through the identification of spectroscopic fingerprints, without the need for fluorescent staining.     

Structural diversity and extreme stability of unimolecular Oxytricha nova telomeric G-quadruplex

Oxytricha nova telomeric DNA contains guanine-rich short-tandem repeat sequences (GGGGTTTT) n and terminates as a single strand at the 3'-end. This single-stranded overhang forms a novel DNA structure, namely, G-quadruplex, comprising four quartets. In this study, we investigated the structures and dynamics of unimolecular Oxytricha nova ( O. nova) telomeric G-quadruplexes by performing single molecule fluorescence resonance energy transfer (FRET) spectroscopy and bulk circular dichroism (CD) measurements. We observed that unimolecular O. nova G-quadruplexes exhibit structural polymorphism according to monovalent cations. In the presence of Na (+), only antiparallel conformation is detected, which was demonstrated in previous studies; however, in the presence of K (+), they fold into two different conformations, a parallel conformation and an antiparallel one different from that induced by Na (+). Furthermore, these G-quadruplexes show extremely high stability in their dynamics when compared with human G-quadruplexes. While human telomeric G-quadruplexes that possess three quartets display fast dynamic behavior (<100 s) at low K (+) concentrations or high temperatures, O. nova G-quadruplexes maintain their conformational state for a long time (>1000 s), even at the lowest K (+) concentration and the highest temperature investigated. This high stability is primarily due to an extra quartet that results in additional cation coordination. In addition to cation coordination, we propose that other factors such as base stacking and the size of the thymine loop may contribute to the stability of O. nova G-quadruplexes; this is based on the fact that the O. nova G-quadruplexes were observed to be more stable than the human ones in the presence of Li (+), which is known to greatly destabilize G-quadruplexes because of imprecise coordination. This extreme stability of four-quartet G-quadruplexes enables telomere protection even in the absence of protective proteins or in the case of abrupt environmental changes, although only a single G-quadruplex structure can be derived from the short single-stranded overhang.     

Interaction between delta and epsilon subunits of F1-ATPase from pig heart mitochondria. Circular dichroism and intrinsic fluorescence of purified and reconstituted delta epsilon complex

A delta epsilon complex has been purified as a molecular entity from pig heart mitochondrial F1-ATPase. This delta epsilon complex has also been reconstituted from purified delta and epsilon subunits. Both isolated and reconstituted delta epsilon complexes have delta 1 epsilon 1 stoichiometry and are indistinguishable by their chromatographic behavior, their circular dichroism spectra (CD spectra), and their intrinsic fluorescence features. The content of secondary structures deduced from CD spectra of the delta epsilon complex appears to be the sum of the respective contributions of purified delta and epsilon subunits. All intrinsic fluorescence studies carried out on isolated epsilon subunit and delta epsilon complex show that the single tryptophan residue located on epsilon is involved in the interaction between delta and epsilon subunits. Results obtained with F1-ATPase are in favor of the same delta epsilon interaction in the entire enzyme.     

Meeting report: Second international meeting on quadruplex DNA

A two and a half day meeting on G-quadruplexes was held in Louisville, KY, USA (April 18–21, 2009). A specific goal of this conference was to promote discussion on the biology of G-quadruplexes. In practice this was represented in four main ways, namely in biophysics, bio/nanotechnology, therapeutics, and what might be termed “intrinsic biology”. Research into the basic biophysical and structural properties of G-quadruplexes continues to be important for understanding biology, and for optimizing aptamers for therapeutic and bio/technological purposes. The meeting comprised two Keynote lectures, twenty-three invited talks, and forty-two posters covering various aspects of these topics using a wide variety of technologies.     

Ligand-induced conformational changes in cytosolic protein kinase C

The changes in intrinsic spectral properties of protein kinase C were monitored upon association with its divalent cation and lipid activators in a model membrane system. The enzyme demonstrated changes in both its intrinsic fluorescence and far ultraviolet circular dichroism spectra upon association with lipid vesicles in the absence of calcium. The acidic phospholipid, phosphatidylserine, significantly quenched the intrinsic tryptophan fluorescence and was also the most potent lipid support for the phosphorylating activity of the enzyme. The enzyme was fully activated by a number of Ca2(+)-lipid combinations which correlated with maximal fluorescence quenching (40-50%) of available tryptophan residues in hydrophobic domains. The circular dichroism structure of the associated active-protein Ca2(+)-lipid complexes suggested different active enzyme secondary structures. However, the Ca2(+)-dependent changes in fluorescence and circular dichroism spectra were observed only after the enzyme associated with the lipid vesicles. These data suggest that protein kinase C has the properties of a complex multidomain protein and provides an additional perspective into the mechanism of protein kinase C activation.     

A stabilizing and denaturing dual-effect for natural polyamines interacting with G-quadruplexes depending on concentration

Both G-quadruplexes and natural polyamines are intimately associated with tumor growth and proliferation. The effect of the natural polyamines on telomeric and some oncogenic G-quadruplexes including bcl-2, c-kit, and c-myc G-quadruplexes has been studied by using absorption, fluorescence, CD, and NMR methods. The results exhibited an interesting dual-effect depending on polyamine? concentration. Polyamines promote and stabilize G-quadruplexes under a lower concentration (less than 1 mM) but denature G-quadruplexes under a higher concentration (more than 1 mM). Probably the electrostatic and hydrophobic effect of polyamines and the hydrogen-bonding interaction between guanines and polyamines were respectively responsible for the stabilizing and denaturing effect.     

Probing the Interaction between Acotiamide Hydrochloride and Pepsin by Multispectral Methods,Electrochemical Measurements,and Docking Studies

The interaction between acotiamide hydrochloride and pepsin was systematically characterized by fluorescence and electrochemical approaches. Fluorescence lifetime measurements showed that acotiamide hydrochloride quenched the intrinsic fluorescence of pepsin with a new complex formation via static mode, which was reconfirmed by cyclic voltammetry results. Both of the binding number and binding constants were calculated from differential pulse voltammetry analysis and fluorescence spectroscopy. The values obtained from the above two methods displayed a relatively high degree of consistency. Thermodynamic parameters suggested that acotiamide hydrochloride interacted with pepsin spontaneously by hydrogen bonding and van der Waals interactions. These results were consistent with the results obtained from molecular docking analysis. As revealed by synchronous fluorescence, three‐dimensional fluorescence, Fourier transform infrared spectrometry, and circular dichroism spectra, acotiamide hydrochloride could affect the microenvironment and slightly change the secondary structure of pepsin. Furthermore, acotiamide hydrochloride can inhibit pepsin activity in vitro, as explained by the molecular docking.     

Two cationic porphyrin isomers showing different multimeric G-quadruplex recognition specificity against monomeric G-quadruplexes

Ligands that can interact specifically with telomeric multimeric G-quadruplexes could be developed as promising anticancer drugs with few side effects related to other G-quadruplex-forming regions. In this paper, a new cationic porphyrin derivative, m-TMPipEOPP, was synthesized and characterized. Its multimeric G-quadruplex recognition specificity under molecular crowding conditions was compared to its isomer p-TMPipEOPP. The slight structural difference accounts for different multimeric G-quadruplex recognition specificity for the two isomers. p-TMPipEOPP can barely discriminate between multimeric and monomeric G-quadruplexes. By contrast, m-TMPipEOPP can bind with multimeric but not with monomeric G-quadruplexes. p-TMPipEOPP might bind to multimeric G-quadruplexes by two modes: sandwich-like end-stacking mode and pocket-dependent intercalative mode. Increasing the pocket size between adjacent two G-quadruplex uints is beneficial for the latter mode. m-TMPipEOPP might bind to multimeric G-quadruplexes by a side binding mode, which confers m-TMPipEOPP with higher multimeric G-quadruplex recognition specificity compared to p-TMPipEOPP. m-TMPipEOPP increases the stability of multimeric G-quadruplex under both dilute and molecular crowding conditions but its G-quadruplex-stabilizing ability is a little weaker than p-TMPipEOPP. These results provide important information for the design of highly specific multimeric G-quadruplex ligands. Another interesting finding is that pocket size is an important factor in determining the stability of multimeric G-quadruplexes.     

Role of loop residues and cations on the formation and stability of dimeric DNA G-quadruplexes

Formation of guanine-quadruplexes by four DNA oligonucleotides with common sequence dG4-loop-dG4 has been studied by a combination of NMR and UV spectroscopy. The loops consisted of 1',2'-dideoxyribose, propanediol, hexaethylene glycol, and thymine residues. The comparison of data on modified and parent oligonucleotides gave insight into the role of loop residues on formation and stability of dimeric G-quadruplexes. All modified oligonucleotides fold into dimeric fold-back G-quadruplexes in the presence of sodium ions. Multiple structures form in the presence of potassium and ammonium ions, which is in contrast to the parent oligonucleotide with dT4 loop. 15N-filtered 1H NMR spectra demonstrate that all studied G-quadruplexes exhibit three 15NH4(+) ion binding sites. Topology of intermolecular G-quadruplexes was evaluated by NMR measurements and diffusion experiments. The spherical, prolate-ellipsoid and symmetric cylinder models were used to interpret experimental translational diffusion constants in terms of diameters and lengths of unfolded oligonucleotides and their respective G-quadruplexes. UV melting and annealing curves show that oligonucleotides with non-nucleosidic loop residues fold faster, exhibit no hysteresis, and are less stable than dimeric d(G4T4G4)2 which can be attributed to the absence of H-bonds, stacking between loop residues and the outer G-quartets as well as cation-pi interactions. Oligonucleotide consisting of hexaethylene glycol linkage with only two phosphate groups in the loop exhibits higher melting temperature and more negative deltaH(o) and deltaG(o) values than oligonucleotides with four 1',2'-dideoxyribose or propanediol residues.     

Insulin-like growth factor type I selectively binds to G-quadruplex structures

Background

G-quadruplex has been viewed as a promising therapeutic target in oncology due to its potentially important roles in physiological and pathological processes. Emerging evidence suggests that the biological functions of G-quadruplexes are closely related to the binding of some proteins. Insulin-like growth factor type I (IGF-1), as a significant modulator of cell growth and development, may serve as a quadruplex-binding protein.

Fluorescence-quenching-resolved spectroscopy of proteins

A new procedure is described for using fluorescence-quenching data of tryptophan residues in proteins to resolve their fluorescence emission spectra. In this concept the Stern-Volmer quenching plot is determined at each particular emission wavelength and iterative non-linear least-squares fitting procedure allowed to resolve the steady-state emission spectra into components. The resolved components, attributed to each of tryptophan residue, can be characterized by different accessibility to the quencher. The ability to resolve fluorescence emission spectra can be improved by using different kinds of efficient quenchers, which can selectively quench the emission of exposed or both exposed and buried fluorophores. The method was used to decompose emission fluorescence spectra in two-tryptophan-containing proteins; horse liver dehydrogenase, sperm whale apomyoglobin and metalloprotease from Staphylococcus aureus. The resolved spectra of alcohol dehydrogenase and metalloprotease are in excellent agreement with those previously obtained by single-photon counting or phase methods. The method presented here is technically simple and does not require expensive instrumentation.