Quadruplex melting

Melting curves are commonly used to determine the stability of folded nucleic acid structures and their interaction with ligands. This paper describes how the technique can be applied to study the properties of four-stranded nucleic acid structures that are formed by G-rich oligonucleotides. Changes in the absorbance (at 295nm), circular dichroism (at 260 or 295nm) or fluorescence of appropriately labelled oligonucleotides, can be used to measure the stability and kinetics of folding. This paper focuses on a fluorescence melting technique, and explains how this can be used to determine the T(m) (T((1/2))) of intramolecular quadruplexes and the effects of quadruplex-binding ligands. Quantitative analysis of these melting curves can be used to determine the thermodynamic (DeltaH, DeltaG, and DeltaS) and kinetic (k(1), k(-1)) parameters. The method can also be adapted to investigate the equilibrium between quadruplex and duplex DNA and to explore the selectivity of ligands for one or other structure.     

PNA-DNA Chimeras Forming Quadruplex Structures

Abstract

¹H-NMR, CD, and UV spectroscopy have been used to investigate the structure of PNA/DNA chimeras forming quadruplex structures. In particular, we synthesized ^5′TGGG^3′-t (1) and ^5′TGG^3′-gt (2), where lower and upper case letters indicate PNA and DNA residues, respectively. CD spectrum and all NMR data of (1) are typical of quadruplexes involving four parallel strands. UV melting profile of (1) indicates that its thermal stability is quite similar to that observed for the reference structure [d(TGGGT)]₄. ¹H-NMR spectrum for ^5′TGG^3′-gt (2) shows that this oligonucleotide is not able to fold into a single, well-defined species.     

Quadruplex structures in nucleic acids.

DNA oligonucleotides that have repetitive tracts of guanine bases can form G-quadruplex structures that display an amazing polymorphism. Structures of several new G-quadruplexes have been solved recently that greatly expand the known structural motifs observed in nucleic acid quadruplexes. Base triads, base hexads, and quartets that contain cytosine have recently been identified stacked over the familiar G-quartets. The current status of the diverse array of structural features in quadruplexes is described and used to provide insight into the polymorphism and folding pathways. This review also summarizes recent progress in the techniques used to probe the structures of G-quadruplexes and discusses the role of ion binding in quadruplex formation. Several of the quadruplex structures featured in this review can be accessed in the online version of this review as CHIME representations.     

Quadruplex DNA crystal structures and drug design

Crystallographic studies of G-quadruplex nucleic acids have resulted in a small group of structures to date. Their morphological and detailed conformational features are described here, emphasizing the stability of the G-tetrad core and the flexibility of loops, especially upon ligand binding. Implications for drug design are discussed, in the context of the druggability of both telomeric and non-telomeric quadruplex DNAs.     

Molecular Recognition of Quadruplex DNA by Quinacridine Derivatives

Abstract

The interaction of monomeric and dimeric quinacridines with quadruplex DNA has been investigated using a variety of biophysical methods. Both series of compounds were shown to exhibit a high affinity for the G4 conformation with two equivalent binding sites. As shown from the SPR and dialysis experiments the macrocyclic dimer appears more selective than its monomeric counterpart.     

Quadruplex DNA: sequence, topology and structure

G-quadruplexes are higher-order DNA and RNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential quadruplex sequences have been identified in G-rich eukaryotic telomeres, and more recently in non-telomeric genomic DNA, e.g. in nuclease-hypersensitive promoter regions. The natural role and biological validation of these structures is starting to be explored, and there is particular interest in them as targets for therapeutic intervention. This survey focuses on the folding and structural features on quadruplexes formed from telomeric and non-telomeric DNA sequences, and examines fundamental aspects of topology and the emerging relationships with sequence. Emphasis is placed on information from the high-resolution methods of X-ray crystallography and NMR, and their scope and current limitations are discussed. Such information, together with biological insights, will be important for the discovery of drugs targeting quadruplexes from particular genes.     

Quadruplex priming amplification combined with nicking enzyme for diagnostics

Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and possesses an intrinsic real-time detection mechanism. QPA can be employed as a diagnostic tool for both linear and exponential signal amplification. The linear QPA, which is less prone to background activity characteristics of exponential systems, suffers from low sensitivity. To increase the sensitivity, here we introduce specific probe molecules that are designed for combined activities of Bst 2.0 polymerase and Nt.BstNBI nicking enzyme. The current assay, which is suitable for single-tube isothermal signal amplification, has increased sensitivity of plain linear QPA by three orders of magnitude to levels of low femtomolar concentration of target molecules.     

Destabilization of DNA Guanine Quadruplex Structure by Foldback Triplex-Forming Oligodeoxynucleotides

Abstract

An oligodeoxynucleotide designed to bind to a single stranded guanine rich DNA sequence through Watson-Crick followed by Hoogsteen hydrogen bonds was found to destabilize quadruplex structure formed by the target sequence. However, the conventional antisense and antigene oligonucleotides were unable to destabilize the same quadruplex structure.     

Dominant Driving Forces in Human Telomere Quadruplex Binding-Induced Structural Alterations

Recently various pathways of human telomere (ht) DNA folding into G-quadruplexes and of ligand binding to these structures have been proposed. However, the key issue as to the nature of forces driving the folding and recognition processes remains unanswered. In this study, structural changes of 22-mer ht-DNA fragment (Tel22), induced by binding of ions (K⁺, Na⁺) and specific bisquinolinium ligands, were monitored by calorimetric and spectroscopic methods and by gel electrophoresis. Using the global model analysis of a wide variety of experimental data, we were able to characterize the thermodynamic forces that govern the formation of stable Tel22 G-quadruplexes, folding intermediates, and ligand-quadruplex complexes, and then predict Tel22 behavior in aqueous solutions as a function of temperature, salt concentration, and ligand concentration. On the basis of the above, we believe that our work sets the framework for better understanding the heterogeneity of ht-DNA folding and binding pathways, and its structural polymorphism.     

Quadruplex ligands may act as molecular chaperones for tetramolecular quadruplex formation

G-quadruplexes are a family of four-stranded DNA structures, stabilized by G-quartets, that form in the presence of monovalent cations. Efforts are currently being made to identify ligands that selectively bind to G-quadruplex motifs as these compounds may interfere with the telomere structure, telomere elongation/replication and proliferation of cancer cells. The kinetics of quadruplex–ligands interactions are poorly understood: it is not clear whether quadruplex ligands lock into the preformed structure (i.e. increase the lifetime of the structure by lowering the dissociation constant, k_off) or whether ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing the association constant, k_on. We studied the effect of a selective quadruplex ligand, a bisquinolinium pyridine dicarboxamide compound called 360A, to distinguish these two possibilities. We demonstrated that, in addition to binding to and locking into preformed quadruplexes, this molecule acted as a chaperone for tetramolecular complexes by acting on k_on. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when evaluating the cellular responses to these agents.     

Detection of Cell Carcinogenic Transformation by a Quadruplex DNA Binding Fluorescent Probe

Cancer can be easily treated when found early. A probe capable of detecting cell transformation may increase the success rate of early diagnosis of cancer. In this report we have tested the ability of a fluorescent, quadruplex DNA binding probe, 3,6-bis(1-methyl-4- vinylpyridinium) carbazole diiodide (BMVC), to detect cell transformation in vitro. BMVC was applied to living cells in several different models of cell transformation, and the fluorescence signals of BMVC were measured. The degrees of cell transformation in these models were characterized by alterations in cellular morphological phenotype and subcellular organization. When BMVC probes were applied, the number of BMVC-positive cells increased in accordance with the degree of transformation. BMVC was capable of significantly detecting formation of foci, increased cellular motility, cell proliferation, cell apoptosis, anchorage-independent growth, and increased invasiveness of transformed cells. These results demonstrate the ability of BMVC probes to detect cell transformation and indicate that BMVC is of promise for use as a probe in early cancer detection.     

Effect of Cationic Comb-Type Copolymer on Quadruplex Folding of Human Telomeric DNA

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA) _n in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA) ₃ GGG) and d((GGGTTA)₇GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA) ₃ GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.     

A Parallel Quadruplex DNA Is Bound Tightly but Unfolded Slowly by Pif1 Helicase

DNA sequences that can form intramolecular quadruplex structures are found in promoters of proto-oncogenes. Many of these sequences readily fold into parallel quadruplexes. Here we characterize the ability of yeast Pif1 to bind and unfold a parallel quadruplex DNA substrate. We found that Pif1 binds more tightly to the parallel quadruplex DNA than single-stranded DNA or tailed duplexes. However, Pif1 unwinding of duplexes occurs at a much faster rate than unfolding of a parallel intramolecular quadruplex. Pif1 readily unfolds a parallel quadruplex DNA substrate in a multiturnover reaction and also generates some product under single cycle conditions. The rate of ATP hydrolysis by Pif1 is reduced when bound to a parallel quadruplex compared with single-stranded DNA. ATP hydrolysis occurs at a faster rate than quadruplex unfolding, indicating that some ATP hydrolysis events are non-productive during unfolding of intramolecular parallel quadruplex DNA. However, product eventually accumulates at a slow rate.     

Folding Topology of a Bimolecular DNA Quadruplex Containing a Stable Mini-hairpin Motif within the Diagonal Loop

We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G₃ACGTAGTG₃)₂ containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G₃T₄G₃)₂ dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T₄-loop sequence. A truncated version of the bimolecular quadruplex d(G₂ACGTAGTG₂)₂, with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.     

1H-NMR Study of the Quadruplex [d(TGGGT)]4 Containing a Modified Thymine

Abstract

A NMR structural study of quadruplex [d(TGGGT)]₄ containing a modified thymine is reported. The three dimensional structure of the complex is very similar to those of other parallel stranded quadruplexes. The modified thymines (T*) are able, at least in the minimised structures, to form a tetrad containing extra H-bonds through the hydroxyl groups. Nevertheless, in this new tetrad the modified thymines are slightly open towards the solvent respect to the unmodified T-tetrad.