Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance

Environments surrounding G-rich sequences remarkably affect the conformations of these structures. A proper evaluation system mimicking the crowded environment in a cell with macromolecules should be developed to perform structural and functional studies on G-quadruplexes. In this study, the topology and stability of a G-quadruplex formed by human telomeric repeat sequences were investigated in a macromolecule-crowded environment created by polyethylene glycol 200 (PEG200), tumor cell extract, and Xenopus laevis egg extract. The interactions between small molecules and telomeric G-quadruplexes were also evaluated in the different systems. The results suggested that the actual behavior of G-quadruplex structures in cells extract is quite different from that in the PEG crowding system, and proteins or other factors in extracts might play a very important role in G-quadruplex structures.     

Specific stabilization of DNA G-quadruplex structures with a chemically modified complementary probe

The DNA G-quadruplex is an important higher-order structure formed from guanine-rich DNA sequences. There are many molecules which can stabilize this structure. However, the selectivity of these ligands to different G-quadruplexes was not satisfactory. Herein, we designed and synthesized a chemically modified G-quadruplex probe, Razo-DNA, for the unique stabilization of the G-quadruplex. Razo-DNA consists of two fragments: The first is an organic molecular moiety which can stabilize G-quadruplex structures, and the second is a DNA molecule that is complementary with a sequence adjacent to the guanine-rich sequence of targeted DNA. Further studies showed that Razo-DNA could precisely stabilize the targeted DNA G-quadruplex structures in vitro.     

Polymorphism of human telomeric quadruplex structures

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). Compounds that can stabilize the intramolecular DNA G-quadruplexes formed in the human telomeric sequence have been shown to inhibit the activity of telomerase and telomere maintenance, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. Knowledge of intramolecular human telomeric G-quadruplex structure(s) formed under physiological conditions is important for structure-based rational drug design and thus has been the subject of intense investigation. This review will give an overview of recent progress on the intramolecular human telomeric G-quadruplex structures formed in K(+) solution. It will also give insight into the structure polymorphism of human telomeric sequences and its implications for drug targeting.     

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.     

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.     

Stability of telomeric G-quadruplexes

In most eukaryotes, telomeric DNA consists of repeats of a short motif that includes consecutive guanines and may hence fold into G-quadruplexes. Budding yeasts have telomeres composed of longer repeats and show variation in the degree of repeat homogeneity. Although telomeric sequences from several organisms have been shown to fold into G-quadruplexes in vitro, surprisingly, no study has been dedicated to the comparison of G-quadruplex folding and stability of known telomeric sequences. Furthermore, to our knowledge, folding of yeast telomeric sequences into intramolecular G-quadruplexes has never been investigated. Using biophysical and biochemical methods, we studied sequences mimicking about four repetitions of telomeric motifs from a variety of organisms, including yeasts, with the aim of comparing the G-quadruplex folding potential of telomeric sequences among eukaryotes. G-quadruplex folding did not appear to be a conserved feature among yeast telomeric sequences. By contrast, all known telomeric sequences from eukaryotes other than yeasts folded into G-quadruplexes. Nevertheless, while G(3)T(1-4)A repeats (found in a variety of organisms) and G(4)T(2,4) repeats (found in ciliates) folded into stable G-quadruplexes, G-quadruplexes formed by repetitions of G(2)T(2)A and G(2)CT(2)A motifs (found in many insects and in nematodes, respectively) appeared to be in equilibrium with non-G-quadruplex structures (likely hairpin-duplexes).     

Recognition of different base tetrads by RHAU (DHX36): X-ray crystal structure of the G4 recognition motif bound to the 3′-end tetrad of a DNA G-quadruplex

G-quadruplexes (G4) are secondary structures of nucleic acids that can form in cells and have diverse biological functions. Several biologically important proteins interact with G-quadruplexes, of which RHAU (or DHX36) – a helicase from the DEAH-box superfamily, was shown to bind and unwind G-quadruplexes efficiently. We report a X-ray co-crystal structure at 1.5 Å resolution of an N-terminal fragment of RHAU bound to an exposed tetrad of a parallel-stranded G-quadruplex. The RHAU peptide folds into an L-shaped α-helix, and binds to a G-quadruplex through π-stacking and electrostatic interactions. X-ray crystal structure of our complex identified key amino acid residues important for G-quadruplex-peptide binding interaction at the 3′-end G•G•G•G tetrad. Together with previous solution and crystal structures of RHAU bound to the 5′-end G•G•G•G and G•G•A•T tetrads, our crystal structure highlights the occurrence of a robust G-quadruplex recognition motif within RHAU that can adapt to different accessible tetrads.     

Effects of G-quadruplex topology on translational inhibition by tRNA fragments in mammalian and plant systems in vitro

The folding of tRNA fragments (tRFs) into G-quadruplex structures and the implications of G-quadruplexes in translational inhibition have been studied mainly in mammalian systems. To increase our knowledge of these phenomena, we determined the influence of human and plant tRFs and model G-quadruplexes on translation in rabbit reticulocyte lysate and wheat germ extract. The efficiency of translational inhibition in the mammalian system was strongly associated with the type of G-quadruplex topology. In the plant system, the ability of a small RNA to adopt the G-quadruplex conformation was not sufficient to repress translation, indicating the importance of other structural determinants.     

Accelerated assembly of G-quadruplex structures by a small molecule.

In the presence of alkali cations, notably potassium and sodium, DNA oligomers that possess two G-rich repeats associate into either a tetrameric parallel G-quadruplex or a variety of dimeric antiparallel G-quadruplexes. The formation of such structures is normally a very slow process. Some proteins, such as the beta-subunit of the Oxytricha telomere-binding protein, promote the formation of G-quadruplex structures in a chaperone-like manner. In this report, we present data concerning the role of a perylene derivative, PIPER, in the assembly of G-quadruplex structures as the first example of a small ligand behaving as a driver in the assembly of polynucleotide secondary structures. Gel-shift experiments demonstrate that PIPER can dramatically accelerate the association of a DNA oligomer containing two tandem repeats of the human telomeric sequence (TTAGGG) into di- and tetrameric G-quadruplexes. In so doing, PIPER alters the oligomer dimerization kinetics from second to first order. The presence of 10 microM PIPER accelerates the assembly of varied dimeric G-quadruplexes an estimated 100-fold from 2 microM oligomer. These results imply that some biological effects elicited by G-quadruplex-interactive agents, such as the induction of anaphase bridges, may stem from the propensity such compounds have for assembling G-quadruplexes.     

NMR spectroscopy of G-quadruplexes

G-rich DNA and RNA sequences can form four-stranded structures called G-quadruplexes. Such structures have gained significant interest in the past decade with increasing evidence of their biological role. G-quadruplex structures can be polymorphic and dynamic. NMR spectroscopy has played an important role in G-quadruplex research. Here we review on the application of NMR techniques to study structure, dynamics and interaction of G-quadruplexes.     

Selective recognition of a DNA G-quadruplex by an engineered antibody

Particular guanine rich nucleic acid sequences can fold into stable secondary structures called G-quadruplexes. These structures have been identified in various regions of the genome that include the telomeres, gene promoters and UTR regions, raising the possibility that they may be associated with biological function(s). Computational analysis has predicted that intramolecular G-quadruplex forming sequences are prevalent in the human genome, thus raising the desire to differentially recognize genomic G-quadruplexes. We have employed antibody phage display and competitive selection techniques to generate a single-chain antibody that shows >1000-fold discrimination between G-quadruplex and duplex DNA, and furthermore >100-fold discrimination between two related intramolecular parallel DNA G-quadruplexes. The amino acid sequence composition at the antigen binding site shows conservation within the light and heavy chains of the selected scFvs, suggesting sequence requirements for G-quadruplex recognition. Circular dichroism (CD) spectroscopic data showed that the scFv binds to the prefolded G-quadruplex and does not induce G-quadruplex structure formation. This study demonstrates the strongest discrimination that we are aware of between two intramolecular genomic G-quadruplexes.     

Hunting G-quadruplexes

Whilst DNA spends much of its time in the double-stranded form, frequently in storage, wrapped around histones and packaged as chromatin, it can also form other complex structures, which may play a role in natural regulation and gene control. These alternative structures therefore also present an interesting novel series of targets for artificial intervention, and so may lead to novel therapeutics. In this review, I describe the current understanding of how genomic and bioinformatics studies may be used to understand the roles that one such structure, the four-stranded guanine-rich G-quadruplex, may play in the genome, and outline how these may be considered as targets for intervention. I will also describe recent work looking at RNA G-quadruplexes, and the biological roles they may play.     

Effective detection and separation method for G-quadruplex DNA based on its specific precipitation with Mg(2+)

G-quadruplex is a type of DNA secondary structure formed by specific guanine-rich sequences. Because of their enrichment in functional genomic regions and their biological significance, G-quadruplexes are recognized as significant drug targets for cancer and other diseases. Here, we tested the precipitation efficiency of Mg(2+) for various DNA oligomers, including single-stranded, double-stranded, triplex, hairpin, i-motif, and some reported G-quadruplex DNA. It was found that Mg(2+) could specifically recognize and precipitate G-quadruplex DNA with a particularly high efficiency of close to 100% for G-quadruplex structures with parallel conformation, which provided an inexpensive and convenient method for detecting and separating G-quadruplex DNA from other DNA structures as well as identifying parallel G-quadruplex from other conformational G-quadruplexes. Further experiments with both CD and gel electrophoresis validated the effectiveness of this approach. The structure of the precipitate was characterized using transmission electron microscopy (TEM), and the observed linear precipitate suggested that a polymerization of G-quadruplex DNA was formed through π-π stacking of end to end by the unique large aromatic surface of G-quadruplex.